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Phosphorylation-dependent and -independent pathways of platelet aggregation.

ABSTRACT: We have used the non-specific inhibitor of protein kinases, staurosporine, to investigate the role of protein phosphorylation during aggregation, the mobilization of intracellular Ca2+ (Ca2+)i and intracellular pH (pHi) in thrombin-stimulated platelets. The concentration of staurosporine chosen for these studies, 1 microM, was previously reported to inhibit protein phosphorylation completely but to have no effect on the activation of phospholipase C in thrombin-stimulated human platelets [Watson, McNally, Shipman & Godfrey (1988) Biochem. J. 249, 345-350]. Aggregation induced by phorbol dibutyrate is slow (several minutes) and is inhibited completely by staurosporine. In contrast, aggregation induced by thrombin, platelet-activating factor or ionophore A23187 is rapid (occurs within 60 s), and is slowed, but not inhibited, in the presence of staurosporine. On the other hand, staurosporine causes a small potentiation of the peak [Ca2+]i signal induced by thrombin and a marked increase in the half-life of decay of this signal, but has no effect on pHi. Under conditions designed to prevent an increase in [Ca2+]i (presence of Ni2+ to prevent Ca2+ entry, and depletion of the intracellular Ca2+ stores), aggregation induced by thrombin resembles that by phorbol dibutyrate and is now inhibited completely by staurosporine. Taken together, these results provide evidence for two signalling pathways for aggregation, a relatively rapid phosphorylation-independent route mediated by Ca2+ and a slower, phosphorylation-dependent, pathway mediated by protein kinase C. Since staurosporine slows aggregation induced by thrombin, it appears that under normal conditions these pathways interact synergistically.

SUBMITTER: Watson SP

PROVIDER: S-EPMC1138386 | biostudies-other | 1989 Mar

REPOSITORIES: biostudies-other

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Project description:Nonsurgical bleeding is the most frequent complication of left ventricular assist device (LVAD) support. Supraphysiologic shear rates generated in LVAD causes impaired platelet aggregation, which increases the risk of bleeding. The effect of shear rate on the formation size of platelet aggregates has never been reported experimentally, although platelet aggregation size can be considered to be directly relevant to bleeding complications. Therefore, this study investigated the impact of shear rate and exposure time on the formation size of platelet aggregates, which is vital in predicting bleeding in patients with an LVAD. Human platelet-poor plasma (containing von Willebrand factor, vWF) and fluorochrome-labeled platelets were subjected to a range of shear rates (0-10 000 s-1 ) for 0, 5, 10, and 15 minutes using a custom-built blood-shearing device. Formed sizes of platelet aggregates under a range of shear-controlled environment were visualized and measured using microscopy. The loss of high molecular weight (HMW) vWF multimers was quantified using gel electrophoresis and immunoblotting. An inhibition study was also performed to investigate the reduction in platelet aggregation size and HMW vWF multimers caused by either mechanical shear or enzymatic (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13-ADAMTS13, the von Willebrand factor protease) mechanism under low and high shear conditions (360 and 10 000 s-1 ). We found that the average size of platelet aggregates formed under physiological shear rates of 360-3000 s-1 (200-300 μm2 ) was significantly larger compared to those sheared at >6000 s-1 (50-100 μm2 ). Furthermore, HMW vWF multimers were reduced with increased shear rates. The inhibition study revealed that the reduction in platelet aggregation size and HWM vWF multimers were mainly associated with ADAMTS13. In conclusion, the threshold of shear rate must not exceed >6000 s-1 in order to maintain the optimal size of platelet aggregates to "plug off" the injury site and stop bleeding.

Project description:BackgroundThe family of 4 related protease-activated receptors (PAR-1, 2, 3 & 4) expressed by mammalian cells allow to sense for and react to extracellular proteolytic activity. Since major human bacterial pathogens secret a wide array of protease(-s) we investigated whether they interfere with human PAR function.Methodology/principal findingsSupernatants from cultures of major human bacterial pathogens were assayed for the presence of protease(-s) capable to cleave overexpressed human PAR-1, 2, 3 and 4 reporter constructs. Group A streptococcus (GAS) was found to secret a PAR-1-cleaving protease. Experiments involving genetical and pharmacological gain and loss of function identified streptococcal pyrogenic exotoxin B SpeB as the protease responsible. On the host's side analysis of overexpressed PAR-1 carrying alanine substitutions and deletions showed the amino acid residue leucine44 on PAR-1's extracellular N-terminus to be the only cleavage site. Complementary studies on endogenously expressed PAR-1 using PAR-1 blocking antibodies further supported our conclusion. Through PAR-1 cleavage SpeB efficiently blunted thrombin-induced induction of the ERK-pathway in endothelial cells and prevented platelets aggregation in response to thrombin.Conclusions/significanceOur results identify a novel function of the streptococcal virulence factor SpeB. By cleaving human PAR-1 at the N-terminal amino acid residue leucine44 SpeB rendered endothelial cells unresponsive to thrombin and prevented human platelets from thrombin-induced aggregation. These results suggest that by blunting PAR-1 signaling, SpeB modulates various innate host responses directed against invasive GAS potentially helping the invasive bacteria to escape. This may allow to tailor additional treatments in the future since upon invasion of the blood stream endothelial cells as well as platelets and mononuclear cells respond to PAR-1 agonists aiming to prevent further bacterial dissemination.

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Phosphorylation-dependent and -independent pathways of platelet aggregation.

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