calpain and Thrombosis

Mild to moderate hypothermia (32-35 degrees C) is the first treatment with proven efficacy for postischemic neurological injury. In recent years important insights have been gained into the mechanisms underlying hypothermia's protective effects; in addition, physiological and pathophysiological changes associated with cooling have become better understood.. To discuss hypothermia's mechanisms of action, to review (patho)physiological changes associated with cooling, and to discuss potential side effects.. Review article.. None.. A myriad of destructive processes unfold in injured tissue following ischemia-reperfusion. These include excitotoxicty, neuroinflammation, apoptosis, free radical production, seizure activity, blood-brain barrier disruption, blood vessel leakage, cerebral thermopooling, and numerous others. The severity of this destructive cascade determines whether injured cells will survive or die. Hypothermia can inhibit or mitigate all of these mechanisms, while stimulating protective systems such as early gene activation. Hypothermia is also effective in mitigating intracranial hypertension and reducing brain edema. Side effects include immunosuppression with increased infection risk, cold diuresis and hypovolemia, electrolyte disorders, insulin resistance, impaired drug clearance, and mild coagulopathy. Targeted interventions are required to effectively manage these side effects. Hypothermia does not decrease myocardial contractility or induce hypotension if hypovolemia is corrected, and preliminary evidence suggests that it can be safely used in patients with cardiac shock. Cardiac output will decrease due to hypothermia-induced bradycardia, but given that metabolic rate also decreases the balance between supply and demand, is usually maintained or improved. In contrast to deep hypothermia (

Topics: Acidosis; Apoptosis; Body Temperature Regulation; Brain Edema; Brain Ischemia; Calpain; Critical Care; Epilepsy; Free Radicals; Genes, Immediate-Early; Humans; Hypothermia, Induced; Infections; Inflammation; Ion Pumps; Mitochondria; Reperfusion Injury; Thrombosis; Thromboxane A2

One of the responses of activated platelets to certain stimuli is the shedding of microparticles. Many studies have attempted to characterize the role of microparticles under various clinical situations or experimental conditions. Pathological levels of fluid shear stress may occur in diseased small arteries and arterioles partially obstructed by atherosclerosis or vasospasm and such shear stress may induce the activation and aggregation of circulating platelets. We investigated whether high shear stress could cause both platelet aggregation and shedding of microparticles from the platelet plasma membrane. A cone-plate viscometer was used to apply shear stress and microparticle formation was measured by flow cytometry. It was found that microparticle formation increased as the duration of shear stress increased. Both microparticles and remnant platelets showed procoagulant activity on their surfaces. Investigation of the mechanisms involved in shear-dependent microparticle generation showed that binding of von Willebrand factor to platelet glycoprotein Ib, influx of extracellular calcium, and activation of platelet calpain were required to generate microparticles under high shear stress conditions. Activation of protein kinase C promoted shear-dependent microparticle formation. These findings suggest that local generation of microparticles in atherosclerotic arteries, the site at which pathological levels of shear stress could occur, contributes to arterial thrombosis by providing and expanding a catalytic surface for the coagulation cascade.

Topics: Arteriosclerosis; Calcium; Calpain; Flow Cytometry; Humans; Platelet Activation; Platelet Glycoprotein GPIb-IX Complex; Protein Binding; Protein Kinase C; Stress, Mechanical; Thrombosis; von Willebrand Factor

Topics: Anemia, Sickle Cell; Blood Platelets; Calpain; Humans; Platelet Activation; Thrombosis

Blood clot contraction is driven by traction forces generated by the platelet cytoskeleton that are transmitted to fibrin fibers via the integrin αIIbβ3. Here we show that clot contraction is impaired by inhibitors of the platelet cytosolic protease calpain. We used subtiligase-mediated labeling of amino termini and mass spectrometry to identify proteolytically cleaved platelet proteins involved in clot contraction. Of 32 calpain-cleaved proteins after TRAP stimulation, 14 were cytoskeletal, most prominently talin and vinculin. A complex of talin and vinculin constitutes a mechanosensitive clutch connecting integrins bound to the extracellular matrix with the actin cytoskeleton. Accordingly, we focused on talin and vinculin. Talin is composed of an N-terminal head domain and a C-terminal rod domain organized into a series of 4- and 5-helix bundles. The bundles contain 11 vinculin binding sites (VBSs), each of which is an α-helix packed into a bundle interior and requiring structural rearrangement to initiate vinculin binding. We detected 8 calpain-mediated cleavages in talin, 2 previously identified in unstructured regions and 6 in α-helical regions in proximity to a VBS. There is evidence in vitro that applying mechanical force across talin enables vinculin binding to the talin rod. However, we found that inhibiting platelet cytoskeletal contraction had no effect on talin cleavage, indicating that talin cleavage by calpain in platelets does not require cytoskeleton-generated tensile force. Therefore, it is likely that calpain acts in the later stages of clot retraction through focal adhesion disassembly.

Topics: Binding Sites; Calpain; Fibrin; Humans; Talin; Thrombosis

Prion diseases are neurodegenerative disorders where infectious prion proteins (PrP) accumulate in brain leading to aggregation of amyloid fibrils and neuronal cell death. The amino acid sequence 106-126 from prion proteins, PrP(106-126), is highly amyloidogenic and implicated in prion-induced pathologies. As PrP is known to be expressed in blood following leakage from brain tissue, we sought to investigate its biological effects on human platelets, which have been widely employed as 'peripheral' model for neurons. Our findings suggested that, PrP(106-126) (20μM) induced dramatic 30-fold rise in intracellular calcium (from 105±30 to 3425±525nM) in platelets, which was attributable to influx from extracellular fluid with comparatively less contribution from intracellular stores. Calcium mobilization was associated with 8-10-fold stimulation in the activity of thiol protease calpain that led to partial cleavage of cytoskeleton-associated protein talin and extensive shedding of microparticles from platelets, thus transforming platelets to 'activated' phenotype. Both proteolysis of talin and microparticle release were precluded by calpeptin, a specific inhibitor of calpain. As microparticles are endowed with phosphatidylserine-enriched surface and hence are pro-coagulant in nature, exposure to prion favored a thrombogenic state in the organism.

Topics: Blood Coagulation; Blood Platelets; Calcium; Calcium Signaling; Calpain; Cell-Derived Microparticles; Cells, Cultured; Cytoskeleton; Dipeptides; Humans; Intracellular Space; Peptide Fragments; Platelet Activation; Prion Diseases; Prions; Proteolysis; Talin; Thrombosis

Oxygen-compromised environments, such as high altitude, air travel, and sports, and pathological conditions, such as solid tumors, have been suggested to be prothrombotic. Despite the indispensable role of platelets in thrombus formation, the studies linking hypoxia, platelet reactivity, and thrombus formation are limited. In the present study, platelet proteome/reactivity was analyzed to elucidate the acute hypoxia-induced prothrombotic phenotype. Rats exposed to acute simulated hypoxia (282 torr/8% oxygen) demonstrated a decreased bleeding propensity and increased platelet reactivity. Proteomic analysis of hypoxic platelets revealed 27 differentially expressed proteins, including those involved in coagulation. Among these proteins, calpain small subunit 1, a 28-kDa regulatory component for calpain function, was significantly upregulated under hypoxic conditions. Moreover, intraplatelet Ca(2+) level and platelet calpain activity were also found to be in accordance with calpain small subunit 1 expression. The inhibition of calpain activity demonstrated reversal of hypoxia-induced platelet hyperreactivity. The prothrombotic role for calpain was further confirmed by an in vivo model of hypoxia-induced thrombosis. Interestingly, patients who developed thrombosis while at extreme altitude had elevated plasma calpain activities and increased soluble P-selectin level. In summary, this study suggests that augmented calpain activity is associated with increased incidence of thrombosis under hypoxic environments.

Topics: Adult; Altitude Sickness; Animals; Blood Platelets; Calpain; Disease Models, Animal; Enzyme Activation; Humans; Hypoxia; Male; Platelet Activation; Proteome; Rats; Rats, Sprague-Dawley; Thrombophilia; Thrombosis

We identified a novel neuroprotective compound, 1-methoxyoctadecan-1-ol, from Uncaria sinensis (Oliv.) Havil and investigated its effects and mechanisms in primary cortical neurons and in a photothrombotic ischemic model. In primary rat cortical neurons against glutamate-induced neurotoxicity, pretreatment with 1-methoxyoctadecan-1-ol resulted in significantly reduced neuronal death in a dose-dependent manner. In addition, treatment with 1-methoxyoctadecan-1-ol resulted in decreased neuronal apoptotic death, as assessed by nuclear morphological approaches. To clarify the neuroprotective mechanism of 1-methoxyoctadecan-1-ol, we explored the downstream signaling pathways of N-methyl-D-aspartate receptor (NMDAR) with calpain activation. Treatment with glutamate leads to early activation of NMDAR, which in turn leads to calpain-mediated cleavage of striatal-enriched protein tyrosine phosphatase (STEP) and subsequent activation of p38 mitogen activated protein kinase (MAPK). However, pretreatment with 1-methoxyoctadecan-1-ol resulted in significantly attenuated activation of GluN2B-NMDAR and a decrease in calpain-mediated STEP cleavage, leading to subsequent attenuation of p38 MAPK activation. We confirmed the critical role of p38 MAPK in neuroprotective effects of 1-methoxyoctadecan-1-ol using specific inhibitor SB203580. In the photothrombotic ischemic injury in mice, treatment with 1-methoxyoctadecan-1-ol resulted in significantly reduced infarct volume, edema size, and improved neurological function. 1-methoxyoctadecan-1-ol effectively prevents cerebral ischemic damage through down-regulation of calpain-mediated STEP cleavage and activation of p38 MAPK. These results suggest that 1-methoxyoctadecan-1-ol showed neuroprotective effects through down-regulation of calpain-mediated STEP cleavage with activation of GluN2B-NMDAR, and subsequent alleviation of p38 MAPK activation. In addition, 1-methoxyoctadecan-1-ol might be a useful therapeutic agent for brain disorder such as ischemic stroke.

Topics: Animals; Brain Ischemia; Calpain; Cerebral Cortex; Disease Models, Animal; Enzyme Inhibitors; Fatty Alcohols; Gene Expression Regulation; Glutamic Acid; Imidazoles; Mice; Neurons; Neuroprotective Agents; p38 Mitogen-Activated Protein Kinases; Photochemical Processes; Primary Cell Culture; Protein Tyrosine Phosphatases, Non-Receptor; Pyridines; Rats; Receptors, N-Methyl-D-Aspartate; Signal Transduction; Thrombosis; Uncaria

Topics: Animals; Blood Platelets; Calpain; Humans; Hypoxia; Male; Thrombosis

When platelets are strongly stimulated, a procoagulant platelet subpopulation is formed that is characterized by phosphatidylserine (PS) exposure and epitope modulation of integrin αIIbβ3 or a loss of binding of activation-dependent antibodies. Mitochondrial permeability transition pore (mPTP) formation, which is essential for the formation of procoagulant platelets, is impaired in the absence of cyclophilin D (CypD). Here we investigate the mechanisms responsible for these procoagulant platelet-associated changes in integrin αIIbβ3 and the physiologic role of procoagulant platelet formation in the regulation of platelet aggregation. Among strongly stimulated adherent platelets, integrin αIIbβ3 epitope changes, mPTP formation, PS exposure, and platelet rounding were closely associated. Furthermore, platelet mPTP formation resulted in a decreased ability to recruit additional platelets. In the absence of CypD, integrin αIIbβ3 function was accentuated in both static and flow conditions, and, in vivo, a prothrombotic phenotype occurred in mice with a platelet-specific deficiency of CypD. CypD-dependent proteolytic events, including cleavage of the integrin β3 cytoplasmic domain, coincided closely with integrin αIIbβ3 inactivation. Calpain inhibition blocked integrin β3 cleavage and inactivation but not mPTP formation or PS exposure, indicating that integrin inactivation and PS exposure are mediated by distinct pathways subsequent to mPTP formation. mPTP-dependent alkalinization occurred in procoagulant platelets, suggesting a possible alternative mechanism for enhancement of calpain activity in procoagulant platelets. Together, these results indicate that, in strongly stimulated platelets, mPTP formation initiates the calpain-dependent cleavage of integrin β3 and associated regulatory proteins, resulting in integrin αIIbβ3 inactivation, and demonstrate a novel CypD-dependent negative feedback mechanism that limits platelet aggregation and thrombotic occlusion.

Topics: Animals; Blood Platelets; Calpain; Cyclophilins; Mice; Mice, Knockout; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Peptidyl-Prolyl Isomerase F; Platelet Aggregation; Platelet Glycoprotein GPIIb-IIIa Complex; Proteolysis; Thrombosis

Calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. Gene targeting was used to evaluate the physiological function of mouse calpain-1 and establish that its inactivation results in reduced platelet aggregation and clot retraction potentially by causing dephosphorylation of platelet proteins. Here, we report that calpain-1 null (Capn1-/-) platelets accumulate protein tyrosine phosphatase 1B (PTP1B), which correlates with enhanced tyrosine phosphatase activity and dephosphorylation of multiple substrates. Treatment of Capn1-/- platelets with bis(N,N-dimethylhydroxamido)hydroxooxovanadate, an inhibitor of tyrosine phosphatases, corrected the aggregation defect and recovered impaired clot retraction. More importantly, platelet aggregation, clot retraction, and tyrosine dephosphorylation defects were rescued in the double knockout mice lacking both calpain-1 and PTP1B. Further evaluation of mutant mice by the ferric chloride-induced arterial injury model suggests that the Capn1-/- mice are relatively resistant to thrombosis in vivo. Together, our results demonstrate that PTP1B is a physiological target of calpain-1 and suggest that a similar mechanism may regulate calpain-1-mediated tyrosine dephosphorylation in other cells.

Topics: Animals; Blood Coagulation; Blood Platelets; Calpain; Mice; Mice, Knockout; Platelet Aggregation; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Signal Transduction; Thrombosis; Vanadates

Excessive accumulation of platelets at sites of athero-sclerotic plaque rupture leads to the development of arterial thrombi, precipitating clinical events such as the acute coronary syndromes and ischemic stroke. The major platelet adhesion receptor glycoprotein (GP) IIb-IIIa (integrin alpha(IIb)beta3) plays a central role in this process by promoting platelet aggregation and thrombus formation. We demonstrate here a novel mechanism down-regulating integrin alpha(IIb)beta3 adhesive function, involving platelet factor XIII (FXIII) and calpain, which serves to limit platelet aggregate formation and thrombus growth. This mechanism principally occurs in collagen-adherent platelets and is induced by prolonged elevations in cytosolic calcium, leading to dramatic changes in platelet morphology (membrane contraction, fragmentation, and microvesiculation) and a specific reduction in integrin alpha(IIb)beta3 adhesive function. Adhesion receptor signal transduction plays a major role in the process by sustaining cytosolic calcium flux necessary for calpain and FXIII activation. Analysis of thrombus formation on a type I fibrillar collagen substrate revealed an important role for FXIII and calpain in limiting platelet recruitment into developing aggregates, thereby leading to reduced thrombus formation. These studies define a previously unidentified role for platelet FXIII and calpain in regulating integrin alpha(IIb)beta3 adhesive function. Moreover, they demonstrate the existence of an autoregulatory feedback mechanism that serves to limit excessive platelet accumulation on highly reactive thrombogenic surfaces.

Topics: Animals; Blood Platelets; Calcium; Calpain; Cell Adhesion; Cell Membrane; Collagen; Cytosol; Down-Regulation; Factor XIII; Fluorescent Antibody Technique, Indirect; Humans; Mice; Platelet Glycoprotein GPIIb-IIIa Complex; Signal Transduction; Thrombosis; Time Factors; von Willebrand Factor

Several earlier studies have implicated platelet activation with the pathogenesis of thrombotic stroke. In this report we have studied the changes in membrane physical microenvironment and signal transduction in the platelets obtained from the patients with thrombotic stroke. Aggregation induced by the synthetic agonist thrombin receptor-activating peptide was significantly enhanced (p < 0.001) in the platelets obtained from the patients. Steady-state fluorescence anisotropy measurements using diphenylhexatriene reflected a significant increase in membrane microviscosity from 3.315 (+/- 0.103) in the control to 4.600 (+/- 0.119) in the stroke. Proteins of relative mobilities of 131, 100, 47 and 38 kDa were found to remain phosphorylated on tyrosine in the resting platelets obtained from thrombotic stroke patients while they were not phosphorylated in the control counter-parts. Besides, calpain, a calcium dependent thiol protease present in the platelets, was found to remain active in this disease as reflected from the proteolysis of calpain substrates. Taken together, these data indicated abnormal circulating platelets in the patients ofthrombotic stroke. which could contribute to the etiopathogenesis of this disease.

Topics: Adult; Blood Platelets; Calpain; Cell Membrane; Diphenylhexatriene; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Female; Fluorescence Polarization; Humans; Male; Membrane Fluidity; Middle Aged; Phosphorylation; Phosphotyrosine; Platelet Aggregation; Signal Transduction; Stroke; Thrombosis