ristocetin and Purpura--Thrombotic-Thrombocytopenic

Von Willebrand factor cleaving protease was first identified in 1987 and was further classified several years later as ADAMTS-13 (a disintegrin and metalloproteinase with thrombospondin-1-like domains). Congenital and acquired deficiency of ADAMTS-13 is associated with thrombotic thrombocytopenic purpura (TTP) and other thrombotic microangiopathies (TMAs). Assays for measurement of ADAMTS-13 were developed in the late 1990s, and significant improvements have occurred in the testing protocols to allow them to be performed in routine hemostasis laboratories. This article reviews the original ADAMTS-13 activity assays and those currently available. It also reviews the consistency of results among various methods and discusses the clinical utility of ADAMTS-13 testing in TTP, TMA, and other disease conditions.

Topics: ADAM Proteins; ADAMTS13 Protein; Collagen; Enzyme-Linked Immunosorbent Assay; Humans; Platelet Aggregation; Protein Binding; Purpura, Thrombotic Thrombocytopenic; Ristocetin; Thrombotic Microangiopathies; von Willebrand Factor

It has been shown that β(2) -glycoprotein I (β(2) GPI) interacts with von Willebrand factor (VWF) in a glycoprotein (GP)Ib binding state. Given the presence of active VWF multimers in thrombotic thrombocytopenic purpura (TTP), we speculated that β(2) GPI might play a role in TTP. We found that β(2) GPI plasma levels were significantly lower in acute and remission TTP patients than in normal controls, showing a direct correlation with ADAMTS 13 levels and an inverse correlation with the extent of VWF activation. In vitro flow experiments demonstrated that β(2) GPI can block platelet adhesion to endothelial cell-derived VWF strings. We confirmed the direct binding of β(2) GPI to VWF by surface plasmon resonance, and determined that domain I of β(2) GPI is the binding site of VWF A1 domain. Adhesion of β(2) GPI to erythrocytes and platelets was increased in the presence of active VWF, indicating that β(2) GPI may be cleared from the circulation during TTP episodes together with blood cells. Our findings suggest that β(2) GPI may protect from the effects of hyper-functional VWF by inhibiting its interaction with platelets.

Topics: Antibodies, Monoclonal, Murine-Derived; beta 2-Glycoprotein I; Blood Platelets; Case-Control Studies; Cells, Cultured; Erythrocytes; HEK293 Cells; Human Umbilical Vein Endothelial Cells; Humans; Platelet Aggregation; Purpura, Thrombotic Thrombocytopenic; Ristocetin; 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

Endothelial cells synthesize and secrete von Willebrand factor (VWF) multimers, including unusually large forms (ULVWF), which are usually cleaved into smaller multimers found in normal plasma (P-VWF). Thrombotic thrombocytopenic purpura (TTP) is a microangiopathic disorder characterized by systemic attachment of platelets to inadequately cleaved ULVWF multimers. We have compared ULVWF and P-VWF in their capacity to become immobilized onto surfaces in vitro and their ability to mediate platelet adhesion. We have also used functional assays to directly compare ULVWF forms with purified P-VWF in mediating platelet aggregation in solution. At comparable concentrations, ULVWF is more effectively adsorbed onto glass surfaces than P-VWF and supports increased platelet adhesion. ULVWF is also significantly more potent than P-VWF in mediating both shear-induced platelet aggregation and ristocetin-mediated platelet agglutination.

Topics: Adult; Endothelial Cells; Endothelium, Vascular; Humans; Molecular Weight; Platelet Adhesiveness; Platelet Aggregation; Purpura, Thrombotic Thrombocytopenic; Ristocetin; Stress, Mechanical; von Willebrand Factor

Endothelial microparticles (EMP) released from activated or apoptotic endothelial cells (EC) are emerging as useful markers for detection of EC dysfunction. Our recent observation that EMP carry von Willebrand factor (vWf) led us to investigate their interaction with platelets. EMP were incubated with normal washed platelets in the presence or absence of ristocetin, then platelet aggregates were measured by flow cytometry. In the absence of ristocetin, negligible EMP conjugated with platelets (< 5%) but in the presence of ristocetin (1 mg mL(-1)), EMP induced up to 95% of platelets to aggregate. EMP-platelet interaction was 80% blocked by anti-CD42b, or by 0.1 microm filtration to remove EMP. Platelet aggregates induced by normal plasma or high molecular weight vWf (Humate-P) dissociated 50% within 15-25 min following 1:20 dilution. In contrast, aggregates formed with EMP persisted two- to threefold longer with the same treatment, indicating greater stability. A similar degree of prolongation of dissociation was observed using plasma from thrombotic thrombocytopenic purpura (TTP) patients compared with normal plasma. Addition of EMP to plasma from severe von Willebrand disease restored his ristocetin-induced platelet aggregation. Multimer analysis of vWf on EMP showed unusually large vWf (ULvWf). In summary, EMP carries ULvWf multimers, promote platelet aggregates, and increase the stability of the aggregates thus formed.

Topics: Cells, Cultured; Dimerization; Endothelium, Vascular; Humans; Macromolecular Substances; Platelet Aggregation; Protein Binding; Purpura, Thrombotic Thrombocytopenic; Ristocetin; von Willebrand Diseases; von Willebrand Factor

We have designed a simple test for the early diagnosis and treatment monitoring of thrombotic thrombocytopenic purpura (TTP). We examined plasma from 24 TTP patients and normal plasma using a cone and plate(let) analyser (CPA). Test plasma was mixed with citrated normal whole blood (group O) and subjected to flow at a shear rate of 1800/s. Mixing normal plasma (12.5, 25, 50 or 75 microl) with heterologous normal whole blood (final volume of 200 microl) resulted in a decrease of surface coverage (SC, maximally by 63%) and, to a lesser extent, of average size (AS, maximally by 37%) due to dilution of the blood sample. In contrast, mixing the same quantities of acute TTP plasma with normal blood yielded an increase in both SC (up to 125%) and AS (up to 130%). Increased SC and/or AS were detected in all 15 patients in acute phase and in three out of 14 patients in remission. Following repeated plasmapheresis, the enhanced platelet deposition in five patients with acute TTP returned to almost normal patterns. Mixing plasma from patients with other thrombocytopenic conditions in this way resulted in a decrease in both SC and AS, and did not differ from control subjects. In conclusion, the CPA is a simple and specific laboratory test that can be used for the diagnosis and monitoring of plasma exchange therapy in TTP.

Topics: Acute Disease; Humans; Infant, Newborn; Plasmapheresis; Platelet Adhesiveness; Platelet Aggregation; Platelet Function Tests; Purpura, Thrombotic Thrombocytopenic; Recurrence; Ristocetin; Sensitivity and Specificity

The haemostatic activity of von Willebrand Factor (VWF) is dependent on VWF multimer stability. Fragments, arising from the proteolytic cleavage of the multimers by VWF-cleaving protease, are ineffective in initiating platelet aggregation. We designed a simple method to determine the protease activity, which was expressed as the reduction in the level of ristocetin-induced platelet aggregation between the inactive enzyme and the enzyme when activated by the addition of calcium. Samples from senior women (n = 14) with chronic coronary heart disease (CHD), age-matched control subjects (n = 15) and young healthy individuals (n = 13), as well as patients with either thrombotic thrombocytopenic purpura (TTP) (n = 2) or von Willebrand disease type 2A (VWD-2A) (n = 2), were examined. The lower protease activity observed in the CHD group, compared with the control subjects, indicated that the increased levels of VWF found in CHD relate to impaired enzyme function. The assessment of TTP patients reconfirmed the reduced protease activity previously observed in this disorder. In VWD-2A, normal enzyme function was observed, suggesting that there is an increased sensitivity of the mutated VWF protein to the protease, rather than an increase in the activity or quantity of the enzyme involved in pathogenesis. In summary, the present simplified method efficiently determines VWF protease activity and is suitable for use in laboratories where platelet aggregation analyses can be performed.

Topics: ADAM Proteins; ADAMTS13 Protein; Adolescent; Adult; Aged; Anti-Bacterial Agents; Case-Control Studies; Coronary Disease; Enzyme Activation; Female; Humans; Metalloendopeptidases; Middle Aged; Platelet Aggregation; Purpura, Thrombotic Thrombocytopenic; Ristocetin; Statistics, Nonparametric; von Willebrand Factor

Extensive microvascular platelet aggregation is characteristic of thrombotic thrombocytopenic purpura (TTP). Previous studies have indicated that abnormalities of von Willebrand factor (vWf) are often present in TTP patient plasma. There has not been previously any direct evidence linking these abnormalities to the process of intravascular platelet aggregation in TTP. We used flow cytometry to analyze the binding of vWf to single platelets, and the presence of platelet aggregates, in the blood of 4 children with chronic relapsing (CR) TTP and 5 adults with single episode or recurrent TTP. vWf on the single platelets of CRTTP patients at all time points studied was significantly increased compared to controls, and was increased further as platelet counts decreased to levels below 40,000/microl. The single episode and recurrent adult TTP patients had platelet aggregates in the blood, as well as increased vWf on single platelets, before therapy commenced and thereafter until recovery was in process. In the one unresponsive single episode TTP patient, vWf on single platelets remained elevated, and platelet aggregates persisted, until her death. The platelet alpha-granular protein, P-selectin, was not increased on the single platelets of most TTP blood samples, suggesting that it is vWf from plasma (rather than from alpha-granules) that attaches to platelet surfaces in association with platelet aggregation. These results suggest that vWf-platelet interactions are involved in the platelet clumping process that characterizes TTP.

Topics: Adenosine Diphosphate; Adult; Blood Platelets; Child; Child, Preschool; Female; Humans; Male; Middle Aged; P-Selectin; Platelet Aggregation; Platelet Count; Protein Binding; Purpura, Thrombotic Thrombocytopenic; Recurrence; Ristocetin; von Willebrand Factor

To further define familial infantile thrombotic thrombocytopenic purpura and clarify its pathophysiology, we describe a family with two infants presenting with this rare syndrome.. Complete, but temporary remission followed the transfusion of whole blood in the first sibling and fresh frozen plasma (FFP) in the second. Periodic FFP transfusions have kept the surviving proband in a prolonged clinical remission. The presence of unusually large von Willebrand factor multimers was demonstrated in the proband and the processing activity of these large multimers was found to be normal.. The occurrence of this rare disorder, in siblings who are products of a consanguinous union, suggests an as yet uncharacterized genetic defect.

Topics: Female; Humans; Infant; Purpura, Thrombotic Thrombocytopenic; Ristocetin; von Willebrand Factor

We have previously observed calpain activity (calcium-dependent cysteine protease) in sera from patients with acute thrombotic thrombocytopenic purpura (TTP). The calpain activity was not present following recovery and was not detected in other thrombocytopenic disorders. We postulated that this enzyme could participate in the pathogenesis of TTP. Because other investigators have demonstrated abnormalities of von Willebrand factor (vWF) in patients with TTP, we proposed that calpain might interact with vWF in TTP. To challenge this hypothesis, we measured the binding of untreated and calpain-treated vWF to normal and ADP or calpain activated platelets. Untreated vWF bound in a specific and saturable fashion to activated platelets, but only at low (30 microM) calcium concentrations. Von Willebrand factor did not bind to activated platelets at physiological (2 mM) calcium concentrations. Calpain proteolysis of vWF changed the binding characteristics of the vWF so that it had greatly increased binding to both ADP and calpain activated platelets. The calpain-proteolyzed vWF bound to activated platelets at both low and physiological calcium concentrations, and was capable of causing platelet aggregation. The calpain-proteolyzed vWF bound to the activated platelets via glycoproteins IIb/IIIa as demonstrated by inhibition studies using monoclonal antibodies against glycoproteins IIb/IIIa and Ib. It also had a high binding affinity and was capable of inhibiting the binding of radiolabelled fibrinogen to the activated platelets at physiological calcium concentrations. Calpain also proteolyzed fibrinogen, but the calpain altered fibrinogen had normal platelet reactivity. These studies provide further insight into the pathogenesis of the platelet aggregation of thrombotic thrombocytopenic purpura. Calpain proteolyses vWF and can produce the characteristic loss of large multimers seen on sodium dodecyl sulphate (SDS)-agarose gel electrophoresis. The altered vWF is highly reactive with activated platelets and binds to platelet glycoproteins IIb/IIIa and participates in formation of the platelet aggregates that characterize this disease.

Topics: Binding, Competitive; Blood Platelets; Calpain; Electrophoresis, Polyacrylamide Gel; Fibrinogen; Humans; Platelet Aggregation; Platelet Membrane Glycoproteins; Purpura, Thrombotic Thrombocytopenic; Ristocetin; von Willebrand Factor

To analyze and review von Willebrand factor (vWF) multimeric patterns in patients with single-episode thrombotic thrombocytopenic purpura (TTP), intermittent TTP (episodes at infrequent, irregular intervals), chronic relapsing TTP (episodes at frequent, regular intervals), and the hemolytic-uremic syndrome (HUS).. Platelet-poor plasma samples were obtained in EDTA, citrate, or citrate-hirudin-aprotinin-leupeptin from 36 patients with single-episode TTP, eight patients with intermittent TTP, four patients with chronic relapsing TTP, and 26 patients with HUS. The samples were separated by sodium dodecyl sulfate-agarose gel electrophoresis, overlaid with rabbit 125I-anti-human vWF IgG, and analyzed by autoradiography.. Abnormalities of vWF multimers were found in platelet-poor plasma samples from 31 of 36 found in platelet-poor plasma samples from 31 of 36 patients (86%) at the onset of and during their single TTP episode. vWF multimers larger than those in normal plasma, and similar to vWF forms observed within normal human endothelial cells (unusually large vWF multimers), were demonstrated in 31% of the patients; 19% had either unusually large vWF multimers or a relative decrease in the largest plasma vWF forms in different serial samples; 36% had a relative decrease in the largest plasma vWF forms. These results imply that endothelial cell injury or intense stimulation, along with the attachment of unusually large vWF multimers and the largest plasma vWF forms to platelets, occurred during the single TTP episodes in most patients. Patterns of vWF multimers were normal in 92% of patients with single-episode TTP studied after recovery. All eight patients with intermittent TTP and the four patients with chronic relapsing TTP had unusually large vWF multimers in their plasma between episodes, and these multimers decreased or disappeared during relapses. Of 26 children and adults with HUS, 14 had a relative decrease in the largest plasma vWF multimeric forms and one had unusually large vWF multimers during the episode (vWF multimeric abnormalities in 58% of the patients).. It is probable that vWF was involved in the pathophysiology of TTP in most of these patients with the single-episode, intermittent, or chronic relapsing types of TTP, and in more than 50% of the patients with HUS.

Topics: Adult; Autoradiography; Child; Chronic Disease; Female; Hemolytic-Uremic Syndrome; Humans; Male; Plasma; Platelet Aggregation; Purpura, Thrombotic Thrombocytopenic; Radiography; Recurrence; Ristocetin; von Willebrand Factor

We have investigated the distribution of vWF multimers in blood from the umbilical cord of infants delivered vaginally and by caesarean section, from heel-stick blood collected 1 d post-partum, and from fetuses undergoing evaluation for Rh compatibility. To examine vWF multimers, plasma was separated by electrophoresis on SDS-agarose gels, overlaid with 125I-anti-vWF, and analysed by densitometry of autoradiographs. Neonatal and fetal plasma contained unusually large von Willebrand factor multimers (ULvWFM), not present in normal adult plasma, in shed blood from adults, in maternal plasma at the time of birth, or in plasma from adults deficient in vitamin K-dependent coagulation proteins. We conclude that ULvWFM, similar in size to vWF present in the Weibel Paladie bodies of endothelial cells, the alpha granules of platelets, and the plasma of patients with TTP, is present in the fetal circulation, at birth, and shortly after delivery.

Topics: Antigens; Electrophoresis, Polyacrylamide Gel; Fetal Blood; Humans; Infant, Newborn; Purpura, Thrombotic Thrombocytopenic; Ristocetin; von Willebrand Factor

Many patients with thrombotic thrombocytopenic purpura (TTP) have a platelet aggregating factor in their serum that may be pathologically linked with the disease process. To help characterize the type of platelet aggregation and platelet release induced by the sera from seven TTP patients, we measured the ability of a variety of inhibitors of platelet function as well as the ability of monoclonal antibodies (MoAbs) against platelet glycoproteins to inhibit TTP sera-induced platelet aggregation and release. These results were compared with the ability of the same inhibitors to block platelet aggregation induced by ristocetin, collagen, ADP, thrombin, and IgG-immune complexes. Monoclonal antibody directed against platelet glycoprotein Ib totally inhibited ristocetin-induced aggregation and release but had no effect on aggregation and release induced by the TTP sera or by any of the other platelet agonists. However, the MoAb against glycoproteins IIb/IIIa inhibited aggregation and release caused by TTP sera as well as by collagen, thrombin, and ADP but had no effect on aggregation and release induced by ristocetin. The aggregating activity could be abolished by heparin but not by the serine protease inhibitor PMSF (1 mmol/L). And although monomeric human IgG and purified Fc fragments of IgG inhibited IgG-immune complex-induced aggregation and release, they had no effect on TTP sera-induced aggregation and release nor on aggregation and release induced by any of the other agonists. Consistent with these in vitro studies showing no effect of IgG were the in vivo observations that intravenous (IV) IgG was without effect when administered to three patients with TTP. This study indicates that although a von Willebrand factor (vWF)-rich preparation of cryoprecipitate enhances the in vitro platelet aggregation and release caused by sera from the seven TTP patients we studied, the pathway of aggregation and release is not via platelet glycoprotein Ib. Also the aggregating factor of TTP sera is not neutralized in vitro or in vivo by IgG.

Topics: Adenosine Diphosphate; Antibodies, Monoclonal; Antigen-Antibody Complex; Blood Platelets; Child, Preschool; Collagen; Female; Humans; Immunoglobulin G; Middle Aged; Platelet Aggregation; Platelet Membrane Glycoproteins; Purpura, Thrombotic Thrombocytopenic; Ristocetin; Thrombin; von Willebrand Factor