Dextran sulphate induces fibrinogen receptor activation through a novel Syk-independent PI-3 kinase-mediated tyrosine kinase pathway in platelets

Abstract

In our attempt to find a physiological agonist that activates PAR3 receptors, we screened several coagulation proteases using PAR4 null platelets. We observed that FXIIa and heat inactivated FXIIa, but not FXII, caused platelet aggregation. We have identified a contaminant activating factor in FXIIa preparation as dextran sulfate (DxS), which caused aggregation of both human and mouse platelets. DxS-induced platelet aggregation was unaffected by YM254890, a Gq inhibitor, but abolished by pan-Src family kinase (SFK) inhibitor PP2, suggesting a role for SFKs in this pathway. However, DxS-induced platelet aggregation was unaffected in FcRγ-chain null murine platelets, ruling out the possibility of glycoprotein VI-mediated events. More interesting, OXSI-2 and Go6976, two structurally unrelated inhibitors shown to affect Syk, had only a partial effect on DxS-induced PAC-1 binding. DxS-induced platelet aggregation and intracellular calcium increases were abolished by the pan PI-3 kinase inhibitor LY294002, or an isoform-specific PI-3 kinase β inhibitor TGX-221. Pretreatment of platelets with Syk inhibitors or ADP receptor antagonists had little effect on Akt phosphorylation following DxS stimulation. These results, for the first time, establish a novel tyrosine kinase pathway in platelets that causes fibrinogen receptor activation in a PI-3 kinase-dependent manner without a crucial role for Syk.

Synthetic glycopolymers and natural fucoidans cause human platelet aggregation via PEAR1 and GPIbα

“…Platelet activation by CLEC-2 and GPVI gives rise to robust elevation of intracellular Ca 21 . In contrast, the activation of platelets by dextran sulfate is associated with only a small change in intracellular Ca 21 , 24 which was confirmed in this study (supplemental Figure 2A). Similarly, C13, C329, and fucoidan from F vesiculosus induced a minimal increase in intracellular Ca 21 in contrast to the marked increase induced by convulxin (supplemental Figure 2A).…”

“…These results show that the synthetic glycopolymers and natural fucoidan activate human platelets via a Src-dependent and Syk-independent pathway that is similar to that described for the sulfated glucoside polysaccharide dextran sulfate. 24 Synthetic glycopolymers and natural fucoidan stimulate PI3K/Akt signaling in human platelets…”

“…The von Willebrand factor receptor GPIba has previously been hypothesized as a possible binding target for the sulfated polysaccharide dextran sulfate. 24 In addition, it has been shown that O-linked carbohydrates are of importance in von Willebrand factor-GPIba binding. 32 In the current study, we show that sulfated synthetic glycopolymers bind to the full-length extracellular domain of GPIba, whereas nonsulfated glycopolymers do not ( Figure 7A).…”

Trypsin causes platelet activation independently of known protease-activated receptors

“…To identify a physiological agonist to PAR3, we used PAR4 null murine platelets, which are known to express only PAR3 (18). We tested several proteases (FXa, FVIIa, FXIIf, elastase, plasma kallikrein, a-chymotrypsin, plasmin, thrombin, tryptase, trypsin) and none of the proteases (25 nM) except trypsin activated PAR4 null murine platelets (19). Trypsin was found to activate PAR4 null murine platelets in a concentration-dependent manner (▶ Figure 1 A).…”

Is the endogenous ligand for PEAR1 a proteoglycan: clues from the sea

“…In contrast to human platelets, where PEAR1 is upregulated on the surface upon activation [2], the majority of PEAR1 is already present on the surface membrane of mouse platelets [7]. The investigation of signalling by PEAR1 in mouse platelets is limited to the use of PEAR1-specific antibodies as, unlike in human platelets, dextran sulfate activates platelets in mice via both PEAR1 and CLEC-2 [4] and fucoidan activates mouse platelets primarily through CLEC-2 [3,12]. This species difference may reflect the twenty fold greater level of expression of CLEC-2 in mouse relative to human platelets [6].…”