Thrombosis and von Willebrand Factor.

One of the key players in both hemostasis and thrombosis is von Willebrand factor (vWF), which demonstrates a duality between these two processes. Thrombus is structured by numerous elements, including endothelial cells, platelets, plasma proteins and shear stress alteration. In circulation, once a vessel wall is injured, collagen is exposed and platelets attach to the site of injury. Accordingly, vWF mediates adherence of platelets to the damaged vessel wall by binding both to the collagen and platelet receptor. A growing body of data also indicates a role for neutrophil extracellular traps (NETs) in human thrombosis as scaffolds for vWF, promoting thrombosis. VWF also mediates the protection of factor VIII, a main cofactor of the intrinsic clotting pathway. Since vWF plays a critical role in both thrombotic and bleeding events, a decreased plasma level of this factor may point to a bleeding disorder, while an elevated plasma level may predict occurrence of thrombosis. Since thrombotic events are the foremost cause of death, inhibiting the vWF activity would be a novel prophylaxis to reduce these events. Though, accumulated data have made vWF a promising focus for research on cardiovascular diseases (CVD). This chapter, however, aims to clarify the role of vWF in thrombus formation and pathogenesis of thrombosis.

Deubiquitinases Modulate Platelet Proteome Ubiquitination, Aggregation, and Thrombosis.

OBJECTIVE: Platelets express a functional ubiquitin-proteasome system. Mass spectrometry shows that platelets contain several deubiquitinases, but whether these are functional, modulate the proteome, or affect platelet reactivity are unknown. APPROACH AND RESULTS: Platelet lysates contained ubiquitin-protein deubiquitinase activity hydrolyzing both Lys48 and Lys63 polyubiquitin conjugates that was suppressed by the chemically unrelated deubiquitinase inhibitors PYR41 and PR619. These inhibitors acutely and markedly increased monoubiquitination and polyubiquitination of the proteome of resting platelets. PYR41 (intravenous, 15 minutes) significantly impaired occlusive thrombosis in FeCl3-damaged carotid arteries, and deubiquitinase inhibition reduced platelet adhesion and retention during high shear flow of whole blood through microfluidic chambers coated with collagen. Total internal reflection microscopy showed that adhesion and spreading in the absence of flow were strongly curtailed by these inhibitors with failure of stable process extension and reduced the retraction of formed clots. Deubiquitinase inhibition also sharply reduced homotypic platelet aggregation in response to not only the incomplete agonists ADP and collagen acting through glycoprotein VI but also to the complete agonist thrombin. Suppressed aggregation was accompanied by curtailed procaspase activating compound-1 binding to activated IIb/IIIa and inhibition of P-selectin translocation to the platelet surface. Deubiquitinase inhibition abolished the agonist-induced spike in intracellular calcium, suppressed Akt phosphorylation, and reduced agonist-stimulated phosphatase and tensin homolog phosphatase phosphorylation. Platelets express the proteasome-associated deubiquitinases USP14 and UCHL5, and selective inhibition of these enzymes by b-AP15 reproduced the inhibitory effect of the general deubiquitinase inhibitors on ex vivo platelet function. CONCLUSIONS: Remodeling of the ubiquitinated platelet proteome by deubiquitinases promotes agonist-stimulated intracellular signal transduction and platelet responsiveness.

Association of platelet collagen receptor polymorphisms with premature acute myocardial infarction.

The impact of platelet collagen receptor polymorphisms in the pathogenesis of myocardial infarction at young age remains unknown. To determine whether either of the two platelet collagen receptor polymorphisms (GP VI T13254C and GP Ia C807T) was associated with premature acute myocardial infarction. One hundred patients with premature acute myocardial infarction and 100 age-matched controls with normal coronary angiograms were studied. Genotyping was done using PCR followed by restriction fragment length polymorphism (RFLP). GP Ia C807T polymorphism was more frequent in the patient group (65%) than in the control group (53%). However, there was no association between this polymorphism and premature acute myocardial infarction (P = 0.08). The prevalence of T13254C polymorphism did not differ between patients (38%) and controls (33%), and this polymorphism was not associated with premature acute myocardial infarction (P = 0.46). Logistic regression analysis also indicated no association between these polymorphisms and premature acute myocardial infarction (C807T with P = 0.51 and T13254C with P = 0.20). There is no association between GP VI T13254C or GP Ia C807T polymorphisms and premature acute myocardial infarction.

Increase in platelet non-integrin type I collagen receptor in patients with systemic sclerosis.

Microvascular injury is one of the major pathogenetic processes involved in systemic sclerosis (SSc). Interaction of the platelet types I and III collagen receptors with their respective ligand in the exposed subendothelial stroma as a result of ongoing microvascular injury in SSc patients results in platelet activation and aggregation with the release of mediators, which contribute to vascular damage and inflammation. We have found that there is a twofold increase in radiolabeled type I collagen binding to washed platelets from patients with SSc compared to platelets obtained from normal volunteers. Western blot analyses showed that the non-integrin platelet type I collagen receptor protein (65 kDa) is increased dramatically in lysates of platelet from patients with SSc. However, the integrin (alpha(2)beta(1)) and other non-integrin receptors such as glycoprotein VI, glycoprotein IV, and the platelet receptor for type III collagen remain unchanged. In addition, platelet lysates from rheumatic disease controls (rheumatoid arthritis, osteoarthritis, gout, and systemic lupus erythematosus) do not show any significant increases. There is no nitrotyrosylation on 65 kDa in patients with SSc compared to controls, suggesting this might also contribute to binding of CI to the 65-kDa CIR. These results suggest that there is a specific increase in the number of platelet type I collagen receptors in SSc patient's platelets. In addition, the activity of nitric oxide synthase is decreased in patients' platelet lysates compared to controls. The increase in platelet expression of the 65-kDa non-integrin platelet type I collagen receptor may explain the enhanced aggregation of platelets from patients with SSc to CI in vitro and microvascular thrombosis in the disease in vivo.

Density of platelet collagen receptors glycoprotein VI and alpha2beta1 and prior myocardial infarction in human subjects, a pilot study.

BACKGROUND: Prior studies evaluating whether platelet collagen receptors affect risk of myocardial infarction (MI) have used common polymorphisms thought to affect receptor density, and have yielded conflicting results. Our objective in the current pilot study was to obtain preliminary information on the relationship between platelet collagen receptor density and prior MI. MATERIAL/METHODS: We measured glycoprotein VI (GPVI) and integrin alpha2beta1 density by flow cytometry using FITC-conjugated monoclonal antibodies in consecutive subjects from an outpatient cardiology practice. We used a case-control design to match 77 patients with prior MI to 77 control patients. Matching was performed on the basis of traditional risk factors for MI. RESULTS: The mean GPVI density was 14% higher in those with prior MI (P = 0.098), and the alpha2beta1 density was 6% higher in those with prior MI (P = 0.49). A GPVI or alpha2beta1 density within the lowest decile was associated with a lower MI prevalence (OR = 6.3, 95% CI: 1.3-29.2 P = 0.009). CONCLUSIONS: Our preliminary data suggests that platelet collagen receptor density, when measured directly, may be related to prior myocardial infarction. An appropriately sized clinical study using this methodology is warranted to further investigate whether collagen receptor density serves as a novel risk factor for myocardial infarction.

Surface expression of collagen receptor Fc receptor-gamma/glycoprotein VI is enhanced on platelets in type 2 diabetes and mediates release of CD40 ligand and activation of endothelial cells.

Diabetes is associated with an enhanced collagen-mediated platelet activation that contributes significantly to thromboischemic complications. In this study, the platelet collagen receptor glycoprotein VI (GPVI) was studied in 385 patients with type 2 diabetes. Surface expression of the platelet Fc receptor that forms a functional complex with GPVI was significantly increased in patients with diabetes compared with those without diabetes (P = 0.02). Fc receptor expression correlated with GPVI expression and was found to be independently associated with diabetes (r = 0.529, P < 0.001). Stimulation of GPVI through a specific anti-GPVI monoclonal antibody significantly enhanced surface expression of CD40L (P = 0.006). Because CD40L is a potent platelet-derived cytokine that is involved in thrombosis and atherosclerosis, we evaluated the effect of GPVI-mediated release of CD40L on activation of endothelial cells. Coincubation of GPVI-stimulated platelets resulted in substantial enhanced endothelial surface expression of CD62P, alphavbeta3, and intercellular adhesion molecule 1 (P < 0.05) and secretion of monocyte chemoattractant protein 1 of cultured human umbilical vein endothelial cells (P < 0.01). These results suggest that the function of collagen receptor GPVI is altered in type 2 diabetes and may play an important role in atherothrombotic complications. Inhibition of GPVI may be a promising pharmacological target in the treatment of high-risk diabetic patients.

Current treatment of von Willebrand's disease.

During the last couple of decades improved pathophysiological insight, use of improved diagnostic tools, and improved understanding of treatment requirements, have converged on an improved safety of treatment and quality of life for the patient suffering VWD. The scientific development in this area has elucidated a vast heterogeneity in VWD now appreciated as a vastly heterogeneous group of bleeding disorders. In some subtypes, dysfunctional VWF protein characteristics are clarified and logically linked with the distinct site of the VWF gene mutation abolishing specific functions of VWF, while in other subtypes such relationships have not yet been established. With the most frequently occurring variant, designated type 1, the quantitative loss in VWF seems to correlate with its reduced function. However, we are only at the beginning of the era of molecular genetics of this specific variant. Medical evidence based guidelines by ordinary standards for treatment of VWD do not exist. Therefore, treatment is based on a mechanistic approach and empirical knowledge. The review presented here focuses on the management of bleeding in VWD. Its basis is a mixture of data reported to literature and the authors' personal clinical observations. Our intention is the presentation and discussion of current issues related to VWD as well as the various pharmaceutical treatment options available for patients afflicted with von Willebrand's disease, together with some theoretical thoughts on possible future modalities.