Evaluation of the physiological significance of botrocetin/ von Willebrand factor in vitro signaling

Abstract

Summary. Background: A signaling pathway is difficult, if not impossible, to elucidate in platelets using only in vivo studies. Likewise, the physiological significance of signaling information obtained exclusively from in vitro observations is unknown. Therefore, both in vitro and in vivo experiments are required to establish the physiological significance of a signaling pathway. Objective: To evaluate the physiological significance of signaling data obtained from botrocetin (bt)/von Willebrand factor (VWF)‐stimulated washed platelets. Method: Stable thrombus formation in response to FeCl3‐induced injury of the mouse carotid artery was used to evaluate the physiological significance of signaling data obtained from bt/VWF‐stimulated washed platelets. Results: Syk, PLCγ2, Gαq and P2Y12, but not LAT, were found either to be required for or to affect stable thrombus formation. Prior in vitro studies had demonstrated that LAT is not required for bt/VWF‐induced platelet aggregation in the presence of exogenous fibrinogen. These data provide the first demonstration of the in vivo role for these signaling molecules in GPIb‐dependent/initiated signal transduction and are consistent with the signaling pathway deduced from in vitro studies of bt/VWF‐stimulated washed platelets using metabolic inhibitors and knockout mice. Conclusion: The broad agreement between the in vitro and the in vivo results establish that bt/VWF stimulation of washed platelets can provide physiologically significant glycoprotein Ib‐dependent/initiated signaling data.

Critical role for Syk in responses to vascular injury

“…As shown in Figure 4A, the time to occlusive thrombus formation was prolonged significantly in PRT060318-treated mice (58 Ϯ 16 vs 33 Ϯ 12 minutes; P ϭ .001). In light of the weak thrombotic phenotype associated with Syk deficiency in an FeCl 3 thrombosis model, 34 we studied the contribution of the kinase activity of Syk in an FeCl 3 thrombosis model applied to mesenteric arteries. In this model, PRT060318 was administered as a continuous intravenous infusion and targeted full inhibition (concentration achieved, 9.6 Ϯ 0.9M) of the kinase activity of Syk.…”

LIM kinase-1 selectively promotes glycoprotein Ib-IX–mediated TXA2 synthesis, platelet activation, and thrombosis

“…9,10 Previous studies indicate that GPIb-IX-induced platelet activation requires the sequential activation of the Src family kinase, Lyn, 11,12 Rac1, 13 PI3K/Akt, 14,15 cGMP-dependent protein kinase, 16 and mitogen-activated protein kinases (MAPKs). 17 However, full platelet responses to VWF require amplification signaling mediated through the immunoreceptor tyrosine-based activation motif signaling pathway, involving either the Fc receptor g-chain and/or Fcg receptor IIA, Syk, SLP76, Bruton tyrosine kinase (Btk), and PLCg2, 11,[18][19][20] followed by consequent thromboxane A 2 (TXA 2 ) and TXA 2 -dependent granule secretion of adenosine diphosphate (ADP). 15,21,22 It remains unclear, though, how GPIb-IX signaling leads to TXA 2 generation.…”

Targeting Glycoprotein VI and the Immunoreceptor Tyrosine-Based Activation Motif Signaling Pathway

“…Importantly, early studies using bone marrow chimeric Syk-deficient mice or pharmacological inhibition of Syk in vivo reported grossly normal hemostasis. 73,76,77 However, a direct comparison of Syk-deficient mice with mice lacking CLEC-2 and GPVI in hemostasis models would be helpful in judging the relative safety of both antithrombotic approaches.…”