ristocetin and Thrombosis

The interaction of platelet GPIbα with von Willebrand factor (VWF) is essential to initiate platelet adhesion and thrombosis, particularly under high shear stress conditions. However, no drug targeting GPIbα has been developed for clinical practice. Here we characterized anfibatide, a GPIbα antagonist purified from snake (Deinagkistrodon acutus) venom, and evaluated its interaction with GPIbα by surface plasmon resonance and in silico modeling. We demonstrated that anfibatide interferds with both VWF and thrombin binding, inhibited ristocetin/botrocetin- and low-dose thrombin-induced human platelet aggregation, and decreased thrombus volume and stability in blood flowing over collagen. In a single-center, randomized, and open-label phase I clinical trial, anfibatide was administered intravenously to 94 healthy volunteers either as a single dose bolus, or a bolus followed by a constant rate infusion of anfibatide for 24 h. Anfibatide inhibited VWF-mediated platelet aggregation without significantly altering bleeding time or coagulation. The inhibitory effects disappeared within 8 h after drug withdrawal. No thrombocytopenia or anti-anfibatide antibodies were detected, and no serious adverse events or allergic reactions were observed during the studies. Therefore, anfibatide was well-tolerated among healthy subjects. Interestingly, anfibatide exhibited pharmacologic effects in vivo at concentrations thousand-fold lower than in vitro, a phenomenon which deserves further investigation.Trial registration: Clinicaltrials.gov NCT01588132.

Topics: Animals; Blood Coagulation; Blood Platelets; Crotalid Venoms; Crotalinae; Fibrinolytic Agents; Healthy Volunteers; Humans; Lectins, C-Type; Models, Molecular; Platelet Adhesiveness; Platelet Aggregation; Platelet Count; Platelet Glycoprotein GPIb-IX Complex; Protein Binding; Protein Conformation; Ristocetin; Snake Venoms; Structure-Activity Relationship; Thrombin; Thrombosis; von Willebrand Factor

Granulocyte colony-stimulating factor (G-CSF) stimulates the bone marrow to produce granulocytes and stem cells and is widely used to accelerate neutrophil recovery after chemotherapy. Interestingly, specific G-CSF receptors have been demonstrated not only on myeloid cells, but also on platelets. Data on the effects of G-CSF on platelet function are limited and partly conflicting. The objective of this study was to determine the effect of G-CSF on platelet aggregation and in vivo platelet activation. Seventy-eight, healthy volunteers were enrolled into this randomised, placebo-controlled trial. Subjects received 5 μg/kg methionyl human granulocyte colony-stimulating factor (r-metHuG-CSF, filgrastim) or placebo subcutaneously for four days. We determined platelet aggregation with a whole blood impedance aggregometer with various, clinically relevant platelet agonists (adenosine diphosphate [ADP], collagen, arachidonic acid [AA], ristocetin and thrombin receptor activating peptide 6 [TRAP]). Filgrastim injection significantly enhanced ADP (+40%), collagen (+60%) and AA (+75%)-induced platelet aggregation (all p<0.01 as compared to placebo and p<0.001 as compared to baseline). In addition, G-CSF enhanced ristocetin-induced platelet aggregation (+18%) whereas TRAP-induced platelet aggregation decreased slightly (-14%) in response to filgrastim. While baseline aggregation with all agonists was only slightly but insignificantly higher in women than in men, this sex difference was enhanced by G-CSF treatment, and became most pronounced for ADP after five days (p<0.001). Enhanced platelet aggregation translated into a 75% increase in platelet activation as measured by circulating soluble P-selectin. G-CSF enhances platelet aggregation and activation in humans. This may put patients suffering from cardiovascular disease and cancer at risk for thrombotic events.

Topics: Adenosine Diphosphate; Adolescent; Adult; Arachidonic Acid; Blood Platelets; Cardiovascular Diseases; Female; Granulocyte Colony-Stimulating Factor; Humans; Male; Middle Aged; Neoplasms; P-Selectin; Peptide Fragments; Platelet Aggregation; Recombinant Proteins; Ristocetin; Sex Factors; Thrombosis

Essentials The role of von Willebrand factor (VWF) domains in regulating platelet adhesion was studied in vivo. Multimeric VWF with spacers at the N- and C-terminus of VWF-A1 were systematically tested. N-terminal modified VWF avidly bound platelet GpIbα, causing VWD Type2B like phenotype in mice. Novel anti-D'D3 mAbs suggest that changes at the D'D3-A1 interface may be biologically relevant.. Background Previous ex vivo studies using truncated VWF (von Willebrand factor) suggest that domain-level molecular architecture may control platelet-GpIbα binding function. Objective We determined if this is the case with multimeric VWF in vivo. Methods Full-length human VWF ('hV') was modified with a 22-amino acid mucinous stretch at either the N-terminus of VWF-A1 to create 'hNV' or C-terminus to yield 'hCV'. This extends the physical distance between VWF-A1 and the adjacent domains by ~6 nm. Similar mucin inserts were also introduced into a human-murine chimera ('h[mA1]V') where murine-A1 replaced human-A1 in hV. This yielded 'h[mA1]NV' and 'h[mA1]CV', with N- and C-terminal inserts. The constructs were tested ex vivo and in vivo. Results Mucin insertion at the N-terminus, but not C-terminus, in both types of constructs resulted in >50-fold increase in binding to immobilized GpIbα. N-terminal insertion also resulted in greater shear-induced platelet activation, more thrombus formation on collagen, enhanced platelet accumulation and slower platelet translocation on immobilized VWF in microfluidics assays. Hydrodynamic injection-based expression of h[mA1]NV, but not h[mA1]V or h[mA1]CV, in VWF

Topics: Animals; Blood Platelets; DNA, Complementary; Female; HEK293 Cells; Hemostasis; Humans; Hydrodynamics; Male; Mice; Microfluidics; Mucins; Phenotype; Platelet Activation; Platelet Adhesiveness; Platelet Function Tests; Protein Binding; Protein Domains; Protein Folding; Ristocetin; Shear Strength; Thrombocytopenia; Thrombosis; von Willebrand Factor

Essentials ADAMTS13 requires a substrate-induced conformational change to attain full activity in vitro. The efficacy of wild type ADAMTS13 in models of thrombosis/stroke may be enhanced by pre-activation. A pre-activated ADAMTS13 variant exhibits enhanced proteolysis of platelet agglutinates. This ADAMTS13 variant is protective in a murine model of stroke at a lower dose than WT ADAMTS13. SUMMARY: Background ADAMTS-13 circulates in a closed conformation, only achieving full proteolytic activity against von Willebrand factor (VWF) following a substrate-induced conformational change. A gain-of-function (GoF) ADAMTS-13 variant (R568K/F592Y/R660K/Y661F/Y665F) is conformationally preactivated. Objectives To establish how the hyperactivity of GoF ADAMTS-13 is manifested in experimental models mimicking the occlusive arterial thrombi present in acute ischemic stroke. Methods The ability of GoF ADAMTS-13 to dissolve VWF-platelet agglutinates was examined with an assay of ristocetin-induced platelet agglutination and in parallel-flow models of arterial thrombosis. A murine model of focal ischemia was used to assess the thrombolytic potential of GoF ADAMTS-13. Results Wild-type (WT) ADAMTS-13 required conformational activation to attain full activity against VWF-mediated platelet capture under flow. In this assay, GoF ADAMTS-13 had an EC

Topics: ADAMTS13 Protein; Animals; Arteries; Blood Platelets; Brain Ischemia; CHO Cells; Cricetinae; Cricetulus; Disease Models, Animal; Humans; Mice; Platelet Aggregation; Protein Conformation; Proteolysis; Recombinant Proteins; Ristocetin; Stroke; Thrombosis

Platelet cross-linking during arterial thrombosis involves von Willebrand Factor (VWF) multimers. Therefore, proteolysis of VWF appears promising to disaggregate platelet-rich thrombi and restore vessel patency in acute thrombotic disorders such as ischemic stroke, acute coronary syndrome, or acute limb ischemia.. Experimental models of thrombotic stroke induced by either intra-arterial thrombin injection or ferric chloride application followed by measurement of cerebral blood flow using a combination of laser Doppler flowmetry and MRI were performed to uncover the effects of NAC on arterial thrombi. To investigate the effect of NAC on larger vessels, we also performed ferric chloride-induced carotid artery thrombosis. In vitro experiments were performed to study the molecular bases of NAC thrombolytic effect, including platelet aggregometry, platelet-rich thrombi lysis assays, thromboelastography (ROTEM), and high-shear VWF string formation using microfluidic devices. We also investigated the putative prohemorrhagic effect of NAC in a mouse model of intracranial hemorrhage induced by in situ collagenase type VII injection.. We demonstrated that intravenous NAC administration promotes lysis of arterial thrombi that are resistant to conventional approaches such as recombinant tissue-type plasminogen activator, direct thrombin inhibitors, and antiplatelet treatments. Through in vitro and in vivo experiments, we provide evidence that the molecular target underlying the thrombolytic effects of NAC is principally the VWF that cross-link platelets in arterial thrombi. Coadministration of NAC and a nonpeptidic GpIIb/IIIa inhibitor further improved its thrombolytic efficacy, essentially by accelerating thrombus dissolution and preventing rethrombosis. Thus, in a new large-vessel thromboembolic stroke model in mice, this cotreatment significantly improved ischemic lesion size and neurological outcome. It is important to note that NAC did not worsen hemorrhagic stroke outcome, suggesting that it exerts thrombolytic effects without significantly impairing normal hemostasis.. We provide evidence that NAC is an effective and safe alternative to currently available antithrombotic agents to restore vessel patency after arterial occlusion.

Topics: Acetylcysteine; Animals; Blood Platelets; Chlorides; Disease Models, Animal; Ferric Compounds; Fibrinolytic Agents; Infarction, Middle Cerebral Artery; Male; Mice; Platelet Aggregation; Ristocetin; Thromboembolism; Thrombosis; Tissue Plasminogen Activator; von Willebrand Factor

Thioredoxin (Trx) is an oxidoreductase with important physiological function. Imbalances in the NADPH/thioredoxin reductase/thioredoxin system are associated with a number of pathologies, particularly cancer, and a number of clinical trials for thioredoxin and thioredoxin reductase inhibitors have been carried out or are underway. Due to the emerging role and importance of oxidoreductases for haemostasis and the current interest in developing inhibitors for clinical use, we thought it pertinent to assess whether inhibition of the NADPH/thioredoxin reductase/thioredoxin system affects platelet function and thrombosis. We used small molecule inhibitors of Trx (PMX 464 and PX-12) to determine whether Trx activity influences platelet function, as well as an unbiased proteomics approach to identify potential Trx substrates on the surface of platelets that might contribute to platelet reactivity and function. Using LC-MS/MS we found that PMX 464 and PX-12 affected the oxidation state of thiols in a number of cell surface proteins. Key surface receptors for platelet adhesion and activation were affected, including the collagen receptor GPVI and the von Willebrand factor receptor, GPIb. To experimentally validate these findings we assessed platelet function in the presence of PMX 464, PX-12, and rutin (a selective inhibitor of the related protein disulphide isomerase). In agreement with the proteomics data, small molecule inhibitors of thioredoxin selectively inhibited GPVI-mediated platelet activation, and attenuated ristocetin-induced GPIb-vWF-mediated platelet agglutination, thus validating the findings of the proteomics study. These data reveal a novel role for thioredoxin in regulating platelet reactivity via proteins required for early platelet responses at sites of vessel injury (GPVI and GPIb). This work also highlights a potential opportunity for repurposing of PMX 464 and PX-12 as antiplatelet agents.

Topics: Benzothiazoles; Blood Coagulation Tests; Blood Platelets; Disulfides; Humans; Hydroquinones; Imidazoles; Platelet Activation; Platelet Adhesiveness; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Function Tests; Platelet Membrane Glycoproteins; Receptors, Collagen; Ristocetin; Thioredoxins; Thrombosis; von Willebrand Factor

We report an unusual case of severe abdominal arterial thrombosis in a young woman using oral desmopressin. Only a few cases with cerebrovascular accidents and coronary syndromes have been described so far, which could be attributed to intravenous administration of desmopressin. Because extensive diagnostic and laboratory investigations for (un)common coagulation disorders could not identify an alternative explanation associated with arterial thrombosis, we hypothesise that desmopressin in an oral dose of at least 200 ug once daily must have been sufficient to cause this dramatic vascular complication. Supportive of our hypothesis, we found remarkably high levels of factor VIII activity, Von Willebrand factor (vWF) antigen and vWF ristocetin cofactor activity (268%, 740%, 590% respectively). To the best of the authors' knowledge, this is the first report suggesting a relationship between oral desmopressin use and life-threatening abdominal arterial thrombosis.

Topics: Abdominal Pain; Adult; Blood Coagulation Disorders; Cerebrovascular Disorders; Deamino Arginine Vasopressin; Echocardiography; Factor VIII; Female; Hemostatics; Humans; Ristocetin; Thrombosis; Tomography, X-Ray Computed; von Willebrand Factor

During IVF, circulating estradiol concentrations are strongly increased, and this may have direct effects on hemostasis. Elevated von Willebrand factor levels represent an important risk factor for arterial and venous thrombosis. ADAMTS13, also known as von Willebrand factor-cleaving protease, has an important regulatory function of von Willebrand factor but has not been studied during IVF. Blood was sampled from 31 women at maximal downregulation of estradiol synthesis using gonadotropin-releasing hormone analogues and during high-level stimulation of estradiol synthesis using follicle-stimulating hormone during the first phase of IVF. Von Willebrand factor antigen, von Willebrand factor ristocetin cofactor activity, factor VIII and ADAMTS13 antigen and activity levels in plasma were determined at the time of downregulation and at high-level stimulation. Estradiol increased from a mean of 154 pg/ml at downregulation to 5889 pg/ml at high-level stimulation (range 1620-19 500 pg/ml). Factor VIII increased from 0.96 ± 0.34 to 1.26 ± 0.41 kIU/l (P < 0.001). Von Willebrand factor antigen and activity increased from 0.75 ± 0.22 to 1.06 ± 0.40 kIU/l (P < 0.001) and from 0.83 ± 0.26 to 1.24 ± 0.48 kIU/l (P < 0.001), respectively. ADAMTS13 antigen decreased from 72.2 ± 13.5 to 67.9 ± 9.9% (P < 0.05, P = 0.01) and ADAMTS13 activity from 88.6 ± 18.3 to 80.8 ± 15.7% (P < 0.01). The increments in estradiol and factor VIII during IVF were paralleled by an increase in von Willebrand factor antigen and activity, and a decrease in circulating ADAMTS13 antigen and activity, respectively. This could in part explain why these patients have an increased risk of thrombotic events.

Topics: ADAM Proteins; ADAMTS13 Protein; Adult; Down-Regulation; Estradiol; Factor VIII; Female; Fertilization in Vitro; Follicle Stimulating Hormone; Gonadotropin-Releasing Hormone; Hemostasis; Humans; Risk; Ristocetin; Thrombosis; Up-Regulation; von Willebrand Factor

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease characterized by systemic microvascular thrombosis caused by adhesion of platelets to ultra-large vWF (ULVWF) multimers. These multimers accumulate because of a deficiency of the processing enzyme ADAMTS13. vWF protein forms long multimers from homodimers that first form through C-terminal disulfide bonds and then join through their N termini by further disulfide bonding. N-acetylcysteine (NAC) is an FDA-approved drug that has long been used to treat chronic obstructive lung disease and acetaminophen toxicity and is known to function in the former disorder by reducing mucin multimers. Here, we examined whether NAC could reduce vWF multimers, which polymerize in a manner similar to mucins. In vitro, NAC reduced soluble plasma-type vWF multimers in a concentration-dependent manner and rapidly degraded ULVWF multimer strings extruded from activated ECs. The effect was preceded by reduction of the intrachain disulfide bond encompassing the platelet-binding A1 domain. NAC also inhibited vWF-dependent platelet aggregation and collagen binding. Injection of NAC into ADAMTS13-deficient mice led to the rapid resolution of thrombi produced by ionophore treatment of the mesenteric venules and reduced plasma vWF multimers. These results suggest that NAC may be a rapid and effective treatment for patients with TTP.

Topics: Acetylcysteine; ADAMTS13 Protein; Animals; Anti-Bacterial Agents; Endothelial Cells; Humans; Male; Metalloendopeptidases; Mice; Mice, Inbred C57BL; Mice, Knockout; Plasma; Platelet Aggregation; Protein Multimerization; Purpura, Thrombotic Thrombocytopenic; Ristocetin; Thrombosis; von Willebrand Factor

Serious thrombotic and hemorrhagic problems or even fatalities evoked by either microgravity or hypergravity occur commonly in the world. We recently reported that platelet functions are inhibited in microgravity environments and activated under high-G conditions, which reveals the pathogenesis for gravity change-related hemorrhagic and thrombotic diseases. However, the mechanisms of platelet functional variations under different gravity conditions remain unclear. In this study we show that the amount of filamin A coimmunoprecipitated with GPIbalpha was enhanced in platelets exposed to modeled microgravity and, in contrast, was reduced in 8 G-exposed platelets. Hypergravity induced actin filament formation and redistribution, whereas actin filaments were reduced in platelets treated with modeled microgravity. Furthermore, intracellular Ca2+ levels were elevated by hypergravity. Pretreatment of platelets with the cell-permeable Ca2+ chelator BAPTA-AM had no effect on cytoskeleton reorganization induced by hypergravity but significantly reduced platelet aggregation induced by ristocetin/hypergravity. Two anti-platelet agents, aspirin and tirofiban, effectively reversed the shortened tail bleeding time and reduced the death rate of mice exposed to hypergravity. Furthermore, the increased P-selectin surface expression was obviously reduced in platelets from mice treated with aspirin/hypergravity compared with those from mice treated with hypergravity alone. These data suggest that the actin cytoskeleton reorganization and intracellular Ca2+ level play key roles in the regulation of platelet functions in different gravitational environments. The results with anti-platelet agents not only further confirm the activation of platelets in vivo but also suggest a therapeutic potential for hypergravity-induced thrombotic diseases.

Topics: Actin Cytoskeleton; Adenosine Diphosphate; Animals; Aspirin; Blood Platelets; Calcium; Chelating Agents; Contractile Proteins; Egtazic Acid; Filamins; Hemostasis; Humans; Hypergravity; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Microfilament Proteins; Platelet Activation; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIb-IX Complex; Protein Binding; Ristocetin; Thrombosis; Time Factors; Tirofiban; Tyrosine; Weightlessness Simulation

Von Willebrand disease (VWD)-type 2B originates from a gain-of-function mutation in von Willebrand factor (VWF), resulting in enhanced platelet binding. Clinical manifestations include increased bleeding tendency, loss of large multimers, thrombocytopenia, and circulating platelet aggregates. We developed a mouse model to study phenotypic consequences of VWD-type 2B mutations in murine VWF: mVWF/R1306Q and mVWF/V1316M. Both mutations allow normal multimerization but are associated with enhanced ristocetin-induced platelet aggregation, typical for VWD-type 2B. In vivo expression resulted in thrombocytopenia and circulating aggregates, both of which were more pronounced for mVWF/V1316M. Furthermore, both mutants did not support correction of bleeding time or arterial vessel occlusion in a thrombosis model. They further displayed a 2- to 3-fold reduced half-life and induced a 3- to 6-fold increase in number of giant platelets compared with wild-type VWF. Loss of large multimers was observed in 50% of the mice. The role of ADAMTS13 was investigated by expressing both mutants in VWF/ADAMTS13 double-deficient mice. ADAMTS13 deficiency resulted in more and larger circulating platelet aggregates for both mutants, whereas the full multimer range remained present in all mice. Thus, we established a mouse model for VWD-type 2B and found that phenotype depends on mutation and ADAMTS13.

Topics: ADAMTS13 Protein; Amino Acid Substitution; Animals; Anti-Bacterial Agents; Bleeding Time; Blood Platelets; Disease Models, Animal; Half-Life; Humans; Metalloendopeptidases; Mice; Mice, Mutant Strains; Mutation, Missense; Platelet Aggregation; Protein Multimerization; Ristocetin; Severity of Illness Index; Thrombocytopenia; Thrombosis; von Willebrand Disease, Type 2; von Willebrand Factor

Many serious thrombotic and haemorrhagic diseases or fatalities have been documented in human being exposed to microgravity or hypergravity environments, such as crewmen in space, roller coaster riders, and aircrew subjected to high-G training. Some possible related organs have been examined to explore the mechanisms underlying these gravity change-related diseases. However, the role of platelets which are the primary players in both thrombosis and haemostasis is unknown. Here we show that platelet aggregation induced by ristocetin or collagen and platelet adhesion to von Willebrand factor (VWF) were significantly decreased after platelets were exposed to simulated microgravity. Conversely, these platelet functions were increased after platelets were exposed to hypergravity. The tail bleeding time in vivo was significantly shortened in mice exposed to high-G force, whereas, was prolonged in hindlimb unloaded mice. Furthermore, three of 23 mice died after 15 minutes of -8 Gx stress. Platelet thrombi disseminated in the heart ventricle and blood vessels in the brain, lung, and heart from the dead mice. Finally, glycoprotein (GP) Ibalpha surface expression and its association with the cytoskeleton were significantly decreased in platelets exposed to simulated microgravity, and obviously increased in hypergravity-exposed platelets. These data indicate that the platelet functions are inhibited in microgravity environments, and activated under high-G conditions, suggesting a novel mechanism for gravity change-related haemorrhagic and thrombotic diseases. This mechanism has important implications for preventing and treating gravity change-related diseases, and also suggests that special attentions should be paid to human actions under different gravity conditions.

Topics: Adenosine Diphosphate; Animals; Blood Platelets; Collagen; Cytoskeleton; Hemorrhage; Hemostasis; Hindlimb Suspension; Humans; Hypergravity; Membrane Glycoproteins; Membrane Proteins; Mice; Models, Animal; P-Selectin; Platelet Adhesiveness; Platelet Aggregation; Platelet Glycoprotein GPIb-IX Complex; Ristocetin; Space Flight; Thrombosis; Time Factors; von Willebrand Factor; Weightlessness Simulation

The Fab-fragment of 6B4, a murine monoclonal antibody targeting the human platelet glycoprotein (GP) Ibalpha and blocking the binding of von Willebrand factor (VWF), is a powerful antithrombotic. In baboons, this was without side effects such as bleeding or thrombocytopenia. Recently, we developed a fully recombinant and humanized version of 6B4-Fab-fragment, h6B4-Fab, which maintains its inhibitory capacities in vitro and ex vivo after injection in baboons. We here investigated the antithrombotic properties, the effect on bleeding time and blood loss and initial pharmacokinetics of h6B4-Fab in baboons. The antithrombotic effect of h6B4-Fab on acute platelet-mediated thrombosis was studied in baboons where thrombus formation is induced at an injured and stenosed site of the femoral artery, allowing for cyclic flow reductions (CFRs) which are measured on an extracorporeal femoral arteriovenous shunt. Injection of 0.5 mg/kg h6B4-Fab significantly reduced the CFRs by 80%, whereas two extra injections, resulting in cumulative doses of 1.5 and 2.5 mg/kg, completely inhibited the CFRs. Platelet receptor occupancy, plasma concentrations and effects ex vivo were consistent with what was previously observed. Finally, minimal effects on bleeding time and blood loss, no spontaneous bleeding and no thrombocytopenia were observed. We therefore conclude that h6B4-Fab maintains the antithrombotic capacities of the murine 6B4-Fab, without causing side effects and therefore can be used for further development.

Topics: Acute Disease; Animals; Anti-Bacterial Agents; Bleeding Time; Blood Platelets; Constriction, Pathologic; Disease Models, Animal; Femoral Artery; Hemorrhage; Humans; Immunoglobulin Fab Fragments; Mice; Papio; Platelet Adhesiveness; Platelet Count; Platelet Glycoprotein GPIb-IX Complex; Regional Blood Flow; Ristocetin; Stress, Mechanical; Thrombocytopenia; Thrombosis

Pathology data from the anthrax animal models show evidence of significant increases in vascular permeability coincident with hemostatic imbalances manifested by thrombocytopenia, transient leucopenia, and aggressive disseminated intravascular coagulation. In this study we hypothesized that anthrax infection modulates the activity of von Willebrand factor (VWF) and its endogenous regulator ADAMTS13, which play important roles in hemostasis and thrombosis, including interaction of endothelial cells with platelets. We previously demonstrated that purified anthrax neutral metalloproteases Npr599 and InhA are capable of cleaving a variety of host structural and regulatory proteins. Incubation of human plasma with these proteases at 37 degrees C in the presence of urea as a mild denaturant results in proteolysis of VWF. Also in these conditions, InhA directly cleaves plasma ADAMTS13 protein. Npr599 and InhA digest synthetic VWF substrate FRETS-VWF73. Amino acid sequencing of VWF fragments produced by InhA suggests that one of the cleavage sites of VWF is located at domain A2, the target domain of ADAMTS13. Proteolysis of VWF by InhA impairs its collagen binding activity (VWF:CBA) and ristocetin-induced platelet aggregation activity. In plasma from anthrax spore-challenged DBA/2 mice, VWF antigen levels increase up to 2-fold at day 3 post-infection with toxigenic Sterne 34F(2) strain, whereas VWF:CBA levels drop in a time-dependent manner, suggesting dysfunction of VWF instead of its quantitative deficiency. This conclusion is further supported by significant reduction in the amount of VWF circulating in blood in the ultra-large forms. In addition, Western blot analysis shows proteolytic depletion of ADAMTS13 from plasma of spore-challenged mice despite its increased expression in the liver. Our results suggest a new mechanism of anthrax coagulopathy affecting the levels and functional activities of both VWF and its natural regulator ADAMTS13. This mechanism may contribute to hemorrhage and thrombosis typical in anthrax.

Topics: ADAM Proteins; ADAMTS13 Protein; Animals; Anthrax; Anti-Bacterial Agents; Bacterial Proteins; Blood Platelets; Cell Communication; Collagen; Disease Models, Animal; Disseminated Intravascular Coagulation; Endothelial Cells; Hemostasis; Humans; Leukopenia; Metalloendopeptidases; Metalloproteases; Mice; Plasma; Platelet Aggregation; Protein Binding; Protein Structure, Tertiary; Ristocetin; Spores, Bacterial; Substrate Specificity; Thrombocytopenia; Thrombosis; Time Factors; Urea; von Willebrand Factor

Thrombus formation is initiated by platelets and leads to cardiovascular, cerebrovascular, and peripheral vascular disease, the leading causes of morbidity and mortality in the Western world. A number of antiplatelet drugs have improved clinical outcomes for thrombosis patients. However, their expanded use, especially in surgery, is limited by hemorrhage. Here, we describe an antiplatelet agent that can have its activity controlled by a matched antidote. We demonstrate that an RNA aptamer targeting von Willebrand factor (VWF) can potently inhibit VWF-mediated platelet adhesion and aggregation. By targeting this important adhesion step, we show that the aptamer molecule can inhibit platelet aggregation in PFA-100 and ristocetin-induced platelet aggregation assays. Furthermore, we show that a rationally designed antidote molecule can reverse the effects of the aptamer molecule, restoring platelet function quickly and effectively over a clinically relevant period. This aptamer-antidote pair represents a reversible antiplatelet agent inhibiting a platelet specific pathway. Furthermore, it is an important step towards creating safer drugs in clinics through the utilization of an antidote molecule.

Topics: Aptamers, Nucleotide; Blood Platelets; Humans; Oligonucleotides; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Function Tests; Ristocetin; SELEX Aptamer Technique; Thrombosis; von Willebrand Factor

We used the Gorog Thrombosis Test to analyze the factors influencing the occlusion time, which represents platelet activation and subsequent occlusive thrombus formation, in 132 healthy Japanese volunteers (116 men, 16 women; mean age, 45.0 +/- 12.0 years). The Gorog Thrombosis Test was designed to evaluate platelet aggregation and thrombolytic activity under a high shear stress condition (175 dynes/cm) in a native blood sample in vitro. The mean +/- SD occlusion time was 154.8 +/- 64.7 s (men, 153.4 +/- 64.2 s and women, 165.4 +/- 56.5 s). The occlusion time was inversely correlated with von Willebrand factor ristocetin cofactor activity (VWF:Rco) (r = -0.242, P = 0.0055) and von Willebrand factor antigen (r = -0.230, P = 0.0080). The mean occlusion time in the group with VWF:Rco of at least 170% (137 s) was significantly shorter than that in the group with VWF:Rco less than 170% (156 s, P < 0.05). Platelet counts, other coagulation markers and smoking showed no significant correlations with occlusion time. Red blood cells (r = -0.177, P = 0.0365), hemoglobin (r = -0.191, P = 0.0245) and hematocrit (r = -0.182, P = 0.0329) also showed inverse correlations with the occlusion time. This report is the first to clearly demonstrate the role of von Willebrand factor in the formation of occlusive thrombi in the Gorog Thrombosis Test.

Topics: Adult; Blood Coagulation; Blood Coagulation Tests; Female; Fibrinolysis; Humans; In Vitro Techniques; Male; Middle Aged; Platelet Activation; Platelet Aggregation; Platelet Function Tests; Reference Values; Ristocetin; Stress, Mechanical; Thrombosis; von Willebrand Factor

The reciprocal modulation of platelet and polymorphonuclear leukocyte (PMN) activities is important in the pathogenesis of thrombosis and inflammation. This study investigated how moderate exercise affects shear-induced platelet activation and subsequent PMNs interaction with platelet-related thrombi under shear flow. Sixteen sedentary healthy men engaged in moderate exercise (about 60% VO(2max)) on a bicycle ergometer. Platelet activation, PMNs interaction with surface-adherent platelets, and PMN-dependent inhibition of platelet activation under shear flow were measured both before and immediately after exercise. The results of this study can be summarized as follows: (1) moderate exercise was associated with lower extents of shear-induced platelet adhesion and aggregation, binding of von Willbrand factor (vWF) to platelets, and glyco-protein IIb/IIIa activation and P-selectin expression on platelet than at rest; (2) the velocity and percentage of rolling PMNs increased while the number of PMNs remaining bound to surface-adherent platelets decreased after moderate exercise; (3) although treating the PMNs with oxidized-low density lipoprotein (Ox-LDL) enhanced PMNs interaction with surface-adherent platelets, moderate exercise suppressed the enhancement of platelet-PMN interaction by Ox-LDL; (4) moderate exercise decreased platelet [Ca (2+)](i) elevation induced by ADP and platelet [Ca(2+)](i) levels mediated by PMN and Ox-LDL-treated PMN; and (5) plasma and PMN-derived nitric oxide metabolites and plasma vWF antigen and activity increased after moderate exercise, whereas plasma and platelet-derived soluble P-selectin levels remained unchanged in response to exercise. Therefore, we conclude that moderate-intensity exercise suppresses shear-induced platelet activation and subsequent PMNs adhesion to platelets deposited at sites of vascular injury under flow, thereby reducing the risks of vascular thrombosis and inflammation.

Topics: Blood Platelets; Calcium; Cell Adhesion; Exercise; Humans; Inflammation; Male; Neutrophils; Nitric Oxide; P-Selectin; Platelet Activation; Platelet Adhesiveness; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Binding; Ristocetin; Stress, Mechanical; Thrombosis; Time Factors; Venous Thrombosis; von Willebrand Factor

Thrombus formation is initiated by adhesion of the platelet receptor, glycoprotein (GP) Ib-IX-V complex, to its adhesive ligand, von Willebrand factor (vWf), in the subendothelium or plasma. The vWf-binding domain of GP Ib-IX-V is in the GP Ibalpha subunit of the complex and contains a leucine-rich repeat region. The adhesion of different leucine-rich repeats was studied using optical tweezers in order to determine which ones were critical for the vWf/GP Ibalpha interaction.. Canine GP Ibalpha does not normally bind to human vWf, and thus canine-human GP Ibalpha chimeras were constructed by sequentially replacing human GP Ibalpha structural regions with their canine counterparts. Chinese hamster ovary (CHO) cells, which are frequently used to express platelet GP complexes, were transfected with the chimeric proteins. Optical tweezers (lambda = 830 nm) were used to investigate bond strengths between vWf and different GP Ibalpha canine-human chimeras. Since vWf does not bind GP Ibalpha without high shear stress, the compounds botrocetin and ristocetin were used to induce binding between human vWf and the chimeras.. All human-canine GP Ibalpha chimeras bound to vWf in the presence of botrocetin. Replacement of the N-terminal flanking sequence and the first leucine-rich repeat resulted in lower GP Ibalpha/vWf bond strengths than the wild-type human GP Ibalpha/vWf bond strength (P < 0.05). Chimeras lacking the second leucine-rich repeat did not adhere to vWf with ristocetin acting as modulator.. The N-terminal flanking sequence and the first leucine-rich repeat of GP Ibalpha were found to be important but not necessary for GP Ibalpha to adhere to vWf. The second leucine-rich repeat was found to be critical for GP Ibalpha to bind vWf and could potentially be used in the development of a novel recombinant anti-thrombotic drugs.

Topics: Animals; CHO Cells; Cricetinae; Cricetulus; Crotalid Venoms; Dogs; Hemostasis; Humans; Lasers; Leucine; Platelet Adhesiveness; Platelet Glycoprotein GPIb-IX Complex; Protein Binding; Recombinant Fusion Proteins; Ristocetin; Thrombosis; von Willebrand Factor

Platelet adhesion to the injured vessel wall is essential in haemostasis and thrombosis. This process involves the interaction of the platelet glycoprotein Ib (GPIb) with surface bound von Willebrand factor (vWF). Since synthetic polycationic peptides of the general formula (Arg)n, (Lys)n or (Arg-Lys)n inhibit GPIb-vWF interaction, they were suggested as potential antithrombotics. Protamine sulphate is a highly cationic polypeptide, arginine accounting for approximately 60% of the primary sequence, utilized to neutralize the anticoagulant effect of heparin after cardiac surgery. We have investigated potential effects of protamine sulphate on the function of GPIb-vWF. Addition of protamine sulphate to platelet-rich plasma (PRP), reduced significantly the GPIb-vWF activity as assessed by ristocetin-induced platelet agglutination. When protamine sulphate was added to PRP containing heparin, even at clinically relevant neutralizing doses the GPIb-vWF activity was reduced by 20-30% (p < 0.001). Protamine sulphate in excess of heparin nearly abolished the activity. Furthermore, the direct effect of protamine sulphate on collagen-induced platelet thrombus formation in non-anticoagulated human blood was investigated by employing an ex-vivo parallel-plate perfusion chamber device. Protamine sulphate (200 microg/mL) reduced platelet-collagen adhesion at shear rates of 650 and 2600 sec(-1) by 40% (p< 0.004) and 45% (p < 0.0001), respectively. The corresponding platelet thrombus volumes were concomitantly reduced by 90% (p < 0.006) and 84% (p < 0.05). Our data are questioning the rationale for empirical repetitive protamine sulphate administration when so-called "heparin rebound" after cardiac surgery is suspected, since protamine sulphate in excess of heparin may impair the platelet GPIb-vWF interaction necessary for normal haemostasis.

Topics: Anticoagulants; Blood Coagulation Tests; Blood Flow Velocity; Collagen; Dose-Response Relationship, Drug; Fibrin; Fibrinolytic Agents; Hemagglutination; Heparin; Humans; Platelet Adhesiveness; Platelet Glycoprotein GPIb-IX Complex; Protamines; Receptors, Cell Surface; Ristocetin; Thrombosis; von Willebrand Factor

The role of glycoprotein Ib (GPIb) in platelet adhesion to subendothelium is well established in human species. However, the interaction of GPIb and von Willebrand factor (VWF) in a widely used experimental model in thrombosis research, that of the pig, has not been clearly elucidated. We investigated the differences between human and pig species in the GPIb/VWF axis in several ways.. Standard aggregometry and perfusion studies with circulating blood were applied to isolated platelets or to blood reconstituted with isolated platelets, VWF and red blood cells from the different species. Platelet aggregation to VWF in the presence of either ristocetin or botrocetin was tested.. Human VWF and ristocetin did not agglutinate pig platelets. However, botrocetin was capable of agglutinating pig platelets. In perfusion studies (800 s(-1), 10 min), washed platelets from both species were suspended in albumin solutions containing human VWF (hVWF) or porcine VWF (pVWF) and red blood cells from the corresponding species. Reconstituted blood with high concentrations of pVWF (> or =0.25 U/mL) caused severe thrombocytopenia during the perfusion procedure when added to human platelets. Nevertheless, lower concentrations (< or =0.1 U/mL) promoted the formation of large aggregates. Under our experimental conditions, hVWF poorly supported pig platelet adhesion.. In conclusion, pVWF may support human platelet adhesion and even promote aggregation, while hVWF can only partially facilitate pig platelet adhesion. Minimal concentrations of pVWF could facilitate the interaction of human platelets with subendothelium, increasing their adhesive and aggregating capabilities. Understanding the molecular differences of the GPIb-VWF axis in different species may prove useful for developing therapeutic strategies aimed at preventing excessive platelet deposition on damaged vascular surfaces.

Topics: Animals; Anti-Bacterial Agents; Aorta; Crotalid Venoms; Female; Humans; Male; Perfusion; Platelet Aggregation; Platelet Glycoprotein GPIb-IX Complex; Protein Binding; Rabbits; Ristocetin; Swine; Thrombosis; von Willebrand Factor

Platelet adhesion in arterial blood flow is mainly supported by the platelet receptor glycoprotein (GP) Ib, which interacts with von Willebrand factor (vWF) that is bound to collagen at the site of vessel wall injury. Antibody 6B4 is a monoclonal antibody (MoAb) raised against purified human GPIb. MoAb 6B4 inhibits both ristocetin- and botrocetin-induced, vWF-dependent human platelet agglutination. MoAb 6B4 furthermore blocks shear-induced adhesion of human platelets to collagen I. We studied the antithrombotic effect of this inhibitory murine MoAb 6B4 in a baboon model of arterial thrombosis. When injected into baboons, intact IgG and its F(ab')(2) fragments caused almost immediate thrombocytopenia, whereas injection of the Fab fragments alone did not. Fab fragments were subsequently used to investigate their in vivo effect on platelet deposition onto a thrombogenic device, consisting of collagen-rich, glutaraldehyde-fixed bovine pericardium (0.6 cm(2)), at a wall shear rate ranging from 700 to 1000 s(-1). Baboons were either pretreated with Fabs to study the effect of inhibition on platelet adhesion or treated 6 minutes after placement of the thrombogenic device to investigate the effect on interplatelet cohesion. Pretreatment of the animals with bolus doses ranging from 80 to 640 microgram/kg Fab fragments significantly reduced (111)In-labeled platelet deposition onto the collagen surface by approximately 43% to 65%. Only the highest dose caused a significant prolongation (doubling) of the bleeding time. Ex vivo ristocetin-induced platelet agglutination was equally reduced. Treatment with a bolus of 110 microgram/kg Fab fragments after a thrombus was allowed to form for 6 minutes had no effect on further platelet deposition. We therefore conclude that Fab fragments or derivatives of inhibitory anti-GPIb antibodies may be useful compounds to prevent thrombosis.

Topics: Animals; Antibodies, Monoclonal; Collagen; Humans; Immunoglobulin Fab Fragments; Male; Mice; Papio; Platelet Adhesiveness; Platelet Aggregation Inhibitors; Platelet Count; Platelet Glycoprotein GPIb-IX Complex; Ristocetin; Thrombosis

To experimentally evaluate the role of von Willebrand factor in thrombosis, a monoclonal antibody-712-, directed against the binding domain of von Willebrand factor to the platelet glycoprotein Ib and a control antibody D4H1, directed against the light chain of Factor VII C, were injected into guinea pigs. A model of experimental thrombosis induced by laser in guinea pig mesenteric arteries was used. The antibody 712 induced a dose-dependent reduction of the thrombus formation without affecting the bleeding time and the platelet number. Ex vivo, the ristocetin-induced platelet aggregation was inhibited by the antibody 712 in a dose-dependent manner while the adenosine diphosphate-induced platelet aggregation was not modified. In conclusion, this study showed that antibody-712- has a significant antithrombotic activity without inducing a hemorrhagic state, suggesting that the von Willebrand factor--glycoprotein Ib axis was a promising target for the strategy of a new antithrombotic therapy.

Topics: Animals; Antibodies, Monoclonal; Binding Sites; Fibrinolytic Agents; Guinea Pigs; Lasers; Mesenteric Arteries; Platelet Aggregation; Platelet Glycoprotein GPIb-IX Complex; Ristocetin; Thrombosis; von Willebrand Factor

Platelet adhesion to exposed subendothelium is mediated by platelet receptor glycoprotein Ib and polymeric von Willebrand factor (vWF). To improve the results of coronary arterial thrombolysis, fragments of vWF with enhanced glycoprotein Ib binding competitive with native vWF have been proposed as adjuvants to recombinant tissue-type plasminogen activator (rtPA). We designed a recombinant vWF fragment spanning Ala444 to Asp730 that contains the Arg545Cys mutation (named AR545C) and analyzed its antiplatelet properties in vitro and in vivo. AR545C-platelet interaction was assessed by ristocetin or botrocetin-induced platelet agglutination, or interaction with extracellular matrix under arterial flow conditions. AR545C showed enhanced reactivity with platelet glycoprotein Ib at low concentrations of ristocetin, and 60% bound spontaneously to platelets. AR545C inhibited ristocetin-induced platelet agglutination in a dose-dependent manner, with a concentration necessary to inhibit 50% of agglutination of 0.16+/-0.04 micromol/L. The inhibitory effect of AR545C on rabbit botrocetin-induced platelet agglutination was also dose dependent, with a concentration necessary to inhibit 50% of agglutination of 0.3 to 0.5 micromol/L. AR545C also completely inhibited aggregate formation and decreased the adhesion of platelets to extracellular matrix by 62.5%. The effect of AR545C on thrombolysis with rtPA was evaluated using a modified rabbit femoral thrombosis model. Local injection of AR545C into the thrombosed segment of rabbit femoral artery significantly shortened the time to reperfusion with rtPA (60+/-17.3 versus 103+/-15.2 minutes, P=.05) and significantly prolonged the total patency time (175 versus 21 minutes, P=.04). No significant difference was found in the reperfusion rate or time to reocclusion. AR545C is a potential antithrombotic agent that enhances the thrombolytic effect of rtPA in the rabbit model.

Topics: Animals; Blood Platelets; Drug Therapy, Combination; Extracellular Matrix; Female; Mutation; Peptide Fragments; Platelet Aggregation; Rabbits; Recombinant Proteins; Ristocetin; Thrombolytic Therapy; Thrombosis; Tissue Plasminogen Activator; von Willebrand Factor

1. A murine anti-human vWF monoclonal antibody, AJvW-2, was developed that inhibited the interaction between platelet glycoprotein Ib (GPIb) and von Willebrand factor (vWF) during the ristocetin- (IC50 = 0.7 +/- 0.1 microgram ml-1) and botrocetin- (IC50 = 1.8 +/- 0.3 microgram ml-1) induced aggregation of human platelets. 2. AJvW-2 inhibited the high shear stress (10.8 N m-2) induced aggregation of human platelets dose-dependently with an IC50 = 2.4 +/- 0.3 micrograms ml-1, but had no effect on low shear stress induced platelet aggregation (1.2 N m-2) up to 100 micrograms ml-1. 3. AJvW-2 also inhibited the high shear stress (5.0 N m-2) induced adhesion of human platelets to collagen I with the same efficacy (IC50 = 2.4 +/- 0.3 micrograms ml-1), but no effect at low shear conditions (1.5 N m-2). 4. AJvW-2 inhibited the botrocetin-induced aggregation of platelets from guinea-pig, rat, rabbit, dog and pig at the same concentration range as human platelets; it likewise also inhibited the high shear stress induced aggregation and adhesion to collagen I of guinea-pig platelets. 5. AJvW-2 prevented arterial thrombus formation in guinea-pigs at a dose of 100 micrograms kg-1 without prolonging the template bleeding time, whereas the GPIIb/IIIa antagonists lamifiban mediated inhibition of thrombosis at 1000 micrograms kg-1 was accompanied by a significant prolongation of the bleeding time. 6. These results suggest that AJvW-2 is a potent inhibitor of the GPIb-vWF interaction and a potential novel antithrombotic agent with lower bleeding risk than GPIIb/IIIa antagonists.

Topics: Animals; Anti-Bacterial Agents; Antibodies, Monoclonal; Bleeding Time; Blood Coagulation; Collagen; Crotalid Venoms; Dogs; Fibrinolytic Agents; Guinea Pigs; Hemorrhage; Humans; Mice; Mice, Inbred BALB C; Platelet Adhesiveness; Platelet Aggregation; Platelet Glycoprotein GPIb-IX Complex; Rabbits; Rats; Ristocetin; Thrombosis; von Willebrand Factor

Topics: Animals; Arteriosclerosis; Blood Platelets; Cardiovascular Diseases; Humans; Hyperlipoproteinemias; Liver Cirrhosis; Lysophosphatidylcholines; Phosphatidylcholine-Sterol O-Acyltransferase; Platelet Adhesiveness; Platelet Aggregation; Rabbits; Ristocetin; Thrombosis; von Willebrand Factor

The authors report a case of chronic eosinophilic leukemia. Ristocetin did not aggregate patient's platelets in the absence of bleeding disorder. A possible relationship between arterial thrombosis observed in patients with eosinophilic leukemia and platelet abnormality is evoked.

Topics: Arteries; Blood Platelet Disorders; Cytoplasmic Granules; Diagnosis, Differential; Eosinophils; Hematopoiesis; Heparin Antagonists; Humans; Male; Middle Aged; Myeloproliferative Disorders; Platelet Adhesiveness; Platelet Aggregation; Ristocetin; Thrombosis; von Willebrand Diseases