ALBERT

Description: Studies of the Syk −/− mouse have implicated spleen tyrosine kinase (Syk), a signaling protein with both kinase and scaffolding activities, in platelet signaling following engagement of GPVI and αIIbβ3 by collagen and fibrinogen, respectively. The present study was designed to determine whether specific inhibition of the kinase activity of Syk, without targeting the Syk scaffolding function, affected in vivo arterial thrombosis. In preliminary experiments, blood from wild-type and Syk−/− mice was perfused through collagen-coated capillaries under arterial shear rates to study ex vivo thrombosis. While blood from wild-type mice formed robust thrombi (37±4.7 μm3/μm2), none was observed in Syk−/− mice. Thrombi intermediate in size (16±3.9 μm3/μm2) developed in Syk+/− mice. To achieve specific pharmacological targeting of the kinase activity of Syk, P142-76, a potent (IC50 = 4 nM) and selective Syk kinase inhibitor was utilized. P142-76 was screened against a broad panel of 139 purified kinases at 50 nM. While Syk kinase was inhibited by 92%, all other kinases retained more than 70% of their activity. In washed human platelets, P142-76 inhibited convulxin (CVX)-induced phosphorylation of LAT (linker for activation of T-cells; IC50 = 111 nM) and intracellular calcium increases (IC50 = 31 nM). The GPVI/Syk-specificity of P142-76 activity was confirmed by its inability to inhibit intracellular calcium increases induced by the PAR1 thrombin receptor agonist TRAP. P142-76 also inhibited CVX-induced aggregation of both human washed platelets (IC50 = 87 nM) and platelet-rich plasma (IC50 = 2.5 μM). Considering the controversial data in respect to the participation of GPVI in arterial thrombosis in murine models, the dependence of arterial thrombosis on Syk function was studied in vivo in pigs. Cross-species activity of P142-76 was confirmed in vitro (CVX-induced PRP aggregation IC50= 350 nM; 5 μM P142-76 completely inhibited thrombosis triggered by collagen in the perfusion chamber assay). At a plasma concentration which abolished ex vivo CVX-induced but not ADP-induced pig platelet aggregation, P142-76 significantly inhibited the deposition of [111In]-labeled platelets in a carotid artery crush swine thrombosis model, without compromising primary hemostasis. % aggregation Swine (n=3) Platelet Deposition % inhibition Plasma Conc (ng/ml) Bleed Time (min) Activated Clotting Time (sec) ADP (20 μM) CVX (250 ng/ml) Control Artery 0 0 3±0.9 133±22 100 100 Treated Artery 76±6.5 1343±304 3.5±0.3 130±13 100 0 To clarify further the contribution of the kinase activity of Syk to arterial thrombosis, effects of P142-76 on human blood were evaluated in real time in the collagen-coated perfusion chamber. Low concentrations of P142-76 (0.3 μM) affected thrombus stability, while increasing concentrations (1–5 μM) delayed and then completely inhibited thrombus formation. Furthermore, P142-76 destabilized pre-formed thrombi, indicating a critical role for Syk in conferring strength to platelet-platelet interactions, i.e. αIIbβ3-mediated cohesion. Our data indicate that the kinase activity of Syk acts in arterial thrombosis through at least two distinct mechanisms. First, Syk kinase confers stability to platelet-platelet interactions downstream of αIIbβ3. Second, it initiates thrombus formation on collagen surfaces. This dual activity of the kinase activity of Syk makes it a preferred target for inhibition of arterial thrombosis, as it does not compromise primary hemostasis.

Description: Although current antiplatelet therapies provide potent antithrombotic effects, their efficacy is limited by a heightened risk of bleeding and failure to affect vascular remodeling after injury. New lines of research suggest that thrombosis and hemorrhage may be uncoupled at the interface of pathways controlling thrombosis and inflammation. Here, as one remarkable example, studies using a novel and highly selective pharmacologic inhibitor of the spleen tyrosine kinase Syk [PRT060318; 2-((1R,2S)-2-aminocyclohexylamino)-4-(m-tolylamino)pyrimidine-5-carboxamide] coupled with genetic experiments, demonstrate that Syk inhibition ameliorates both the acute and chronic responses to vascular injury without affecting hemostasis. Specifically, lack of Syk (murine radiation chimeras) attenuated shear-induced thrombus formation ex vivo, and PRT060318 strongly inhibited arterial thrombosis in vivo in multiple animal species while having minimal impact on bleeding. Furthermore, leukocyte-platelet–dependent responses to vascular injury, including inflammatory cell recruitment and neointima formation, were markedly inhibited by PRT060318. Thus, Syk controls acute and long-term responses to arterial vascular injury. The therapeutic potential of Syk may be exemplary of a new class of antiatherothrombotic agents that target the interface between thrombosis and inflammation.

Description: Glycoprotein (GP) VI, the primary collagen receptor on platelets, has been shown to have variable expression, possibly as a consequence of immune modulation. The present study was designed to determine the mechanism by which GP VI clearance occurs. We found that direct activation of GP VI both by a GP VI–specific antibody and by GP VI ligands (collagen and convulxin) reduced binding of biotinylated convulxin to the stimulated platelets. Analysis of immunoblots of platelets and supernatants showed that the stimulated platelets contained less GP VI, while the soluble fraction contained a 57-kDa cleavage product. Stimulation of platelets with PAR-1 agonists (TRAP peptide and thrombin) also caused GP VI cleavage, although the amount of GP VI loss was less than that observed with direct GP VI ligands. The metalloproteinase (MMP) inhibitors GM6001 and TAPI prevented both the clearance of GP VI from the platelet surface and the appearance of the soluble cleavage product. Induction of GP VI cleavage caused specific down-regulation of collagen-induced platelet aggregation, providing a mechanism for the modulation of platelet responsiveness to this important platelet agonist.