Dual-specificity phosphatase 3 deficiency or inhibition limits platelet activation and arterial thrombosis.

BACKGROUND: A limitation of current antiplatelet therapies is their inability to separate thrombotic events from bleeding occurrences. A better understanding of the molecular mechanisms leading to platelet activation is important for the development of improved therapies. Recently, protein tyrosine phosphatases have emerged as critical regulators of platelet function. METHODS AND RESULTS: This is the first report implicating the dual-specificity phosphatase 3 (DUSP3) in platelet signaling and thrombosis. This phosphatase is highly expressed in human and mouse platelets. Platelets from DUSP3-deficient mice displayed a selective impairment of aggregation and granule secretion mediated by the collagen receptor glycoprotein VI and the C-type lectin-like receptor 2. DUSP3-deficient mice were more resistant to collagen- and epinephrine-induced thromboembolism compared with wild-type mice and showed severely impaired thrombus formation on ferric chloride-induced carotid artery injury. Intriguingly, bleeding times were not altered in DUSP3-deficient mice. At the molecular level, DUSP3 deficiency impaired Syk tyrosine phosphorylation, subsequently reducing phosphorylation of phospholipase Cγ2 and calcium fluxes. To investigate DUSP3 function in human platelets, a novel small-molecule inhibitor of DUSP3 was developed. This compound specifically inhibited collagen- and C-type lectin-like receptor 2-induced human platelet aggregation, thereby phenocopying the effect of DUSP3 deficiency in murine cells. CONCLUSIONS: DUSP3 plays a selective and essential role in collagen- and C-type lectin-like receptor 2-mediated platelet activation and thrombus formation in vivo. Inhibition of DUSP3 may prove therapeutic for arterial thrombosis. This is the first time a protein tyrosine phosphatase, implicated in platelet signaling, has been targeted with a small-molecule drug.

Similar hypercoagulable state and thrombosis risk in type I and type III protein S-deficient individuals from families with mixed type I/III protein S deficiency.

BACKGROUND: Protein S, which circulates in plasma in both free and bound forms, is an anticoagulant protein that stimulates activated protein C and tissue factor pathway inhibitor. Hereditary type I protein S deficiency (low total and low free protein S) is a well-established risk factor for venous thrombosis, whereas the thrombosis risk associated with type III deficiency (normal total and low free protein S) has been questioned. DESIGN AND METHODS: Kaplan-Meier analysis was performed on 242 individuals from 30 families with protein S deficiency. Subjects were classified as normal, or having type I or type III deficiency according to their total and free protein S levels. Genetic and functional studies were performed in 23 families (132 individuals). RESULTS: Thrombosis-free survival was not different between type I and type III protein S-deficient individuals. Type III deficient individuals were older and had higher protein S, tissue factor pathway inhibitor and prothrombin levels than type I deficient individuals. Thrombin generation assays sensitive to the activated protein C- and tissue factor pathway inhibitor-cofactor activities of protein S revealed similar hypercoagulable states in type I and type III protein S-deficient plasma. Twelve PROS1 mutations and two large deletions were identified in the genetically characterized families. CONCLUSIONS: Not only type I, but also type III protein S deficiency is associated with a hypercoagulable state and increased risk of thrombosis. These findings may, however, be restricted to type III deficient individuals from families with mixed type I/III protein S deficiency, as these represented 80% of type III deficient individuals in our cohort.

Similar hypercoagulable state and thrombosis risk in type I and type III protein S-deficient individuals from mixed type I/III families.

BACKGROUND: Protein S, which circulates in plasma in a free and bound form, is an anticoagulant protein that stimulates both activated protein C (APC) and tissue factor pathway inhibitor (TFPI). Hereditary type I protein S deficiency (low total and low free protein S) is a well-established risk factor for venous thrombosis, whereas the thrombosis risk associated with type III deficiency (normal total and low free protein S) has been questioned. DESIGN AND METHODS: Kaplan-Meier analysis was performed on 242 individuals from 30 families with protein S deficiency. Subjects were classified as normal, type I deficient or type III deficient according to their total and free protein S levels. Genetic and functional studies were performed in 23 families (132 individuals). RESULTS: Thrombosis-free survival was not different between type I and type III protein S-deficient individuals. Type III deficient individuals were older and had higher protein S, TFPI and prothrombin levels than type I deficient individuals. Thrombin generation assays sensitive to the APC- and TFPI-cofactor activities of protein S revealed similar hypercoagulable states in type I and type III protein S-deficient plasma. Twelve PROS1 mutations and 2 large deletions were identified in the genetically characterized families. CONCLUSIONS: Not only type I, but also type III protein S deficiency is associated with a hypercoagulable state and increased thrombosis risk. However, these findings may be restricted to type III deficient individuals from families with mixed type I/III protein S deficiency, as these represented 80% of type III deficient individuals in our cohort.

A positive family history for premature cardiovascular disease identifies patients prone to recurrent arterial thrombotic events.

BACKGROUND: Cardiovascular disease (CVD) is characterized by slow progressive atherosclerosis and arterial thrombotic events, leading to occlusions. Whether either of these presentations is more likely in patients with a genetic predisposition for CVD is still unknown. We suggest that a genetic predisposition for CVD is related to recurrent events of the same nature. METHODS: We retrospectively investigated 275 patients with premature CVD and divided them in two groups according to their first event: an arterial thrombotic event or stable atherosclerosis. We used a Cox proportional-hazards model to estimate the effect of a positive family history for CVD on recurrent events of the same nature. This was tested in the entire cohort and in patients with coronary artery disease only. RESULTS: Patients with a first arterial thrombotic event and a positive family history had a threefold increased risk for a recurrent event of the same nature, compared to patients with a negative family history (hazard ratio 3.00, 95% confidence interval 1.32-6.81); p < 0.05). In contrast, a positive family history was not associated with an increased risk for a recurrent stable atherosclerosis (hazard ratio 0.98 (95% confidence interval 0.59-1.63). These findings were similar analysing the patients with coronary artery disease only. Additional adjustments for other risk factors did not change these associations. CONCLUSIONS: Patients with a first premature arterial thrombotic event and a positive family history for CVD have an increased risk for a second event of the same nature. This might be due to unknown hereditary mechanisms leading to recurrent acute events.

Re-evaluation of the role of the protein S-C4b binding protein complex in activated protein C-catalyzed factor Va-inactivation.

Protein S expresses cofactor activity for activated protein C (APC) by enhancing the APC-catalyzed proteolysis at R(306) in factor Va. It is generally accepted that only free protein S is active and that complex formation with C4b-binding protein (C4BP) inhibits the APC-cofactor activity of protein S. However, the present study shows that protein S-C4BP expresses APC-cofactor activity and stimulates APC-catalyzed proteolysis at R(306) more than 10-fold, but instead inhibits proteolysis at R(506) by APC 3- to 4-fold. Free protein S stimulates APC-catalyzed cleavage at R(306) approximately 20-fold and has no effect on cleavage at R(506). The resulting net effect of protein S-C4BP complex formation on APC-catalyzed factor Va inactivation is a 6- to 8-fold reduction in factor Va inactivation when compared with free protein S, which is not explained by inhibition of APC-cofactor activity of protein S at R(306), but by generation of a specific inhibitor for APCcatalyzed proteolysis at R(506) of factor Va. These results are of interest for carriers of the factor V(Leiden) mutation (R(506)Q), as protein S-C4BP effectively enhances APC-catalyzed factor Va (R(306)) inactivation in plasma containing factor V(Leiden).