Protease-activated receptors and glycoprotein VI cooperatively drive the platelet component in thromboelastography - PubMed (original) (raw)

Protease-activated receptors and glycoprotein VI cooperatively drive the platelet component in thromboelastography

Tanvi Rudran et al. J Thromb Haemost. 2023 Aug.

Abstract

Background: Thromboelastography (TEG) is used for real-time determination of hemostatic status in patients with acute risk of bleeding. Thrombin is thought to drive clotting in TEG through generation of polymerized fibrin and activation of platelets through protease-activated receptors (PARs). However, the specific role of platelet agonist receptors and signaling in TEG has not been reported.

Objectives: Here, we investigated the specific receptors and signaling pathways required for platelet function in TEG using genetic and pharmacologic inhibition of platelet proteins in mouse and human blood samples.

Methods: Clotting parameters (R time, α-angle [α], and maximum amplitude [MA]), were determined in recalcified, kaolin-triggered citrated blood samples using a TEG 5000 analyzer.

Results: We confirmed the requirement of platelets, platelet contraction, and αIIbβ3 integrin function for normal α and MA. Loss of the integrin adaptor Talin1 in megakaryocytes/platelets (Talin1mKO) also reduced α and MA, but only minimal defects were observed in samples from mice lacking Rap1 GTPase signaling. PAR4mKO samples showed impaired α but normal MA. However, impaired TEG traces similar to those in platelet-depleted samples were observed with samples from PAR4mKO mice depleted of glycoprotein VI on platelets or with addition of a Syk inhibitor. We reproduced these results in human blood with combined inhibition of PAR1, PAR4, and Syk.

Conclusion: Our results demonstrate that standard TEG is not sensitive to platelet signaling pathways critical for integrin inside-out activation and platelet hemostatic function. Furthermore, we provide the first evidence that PARs and glycoprotein VI play redundant roles in platelet-mediated clot contraction in TEG.

Keywords: bleeding; blood platelets; fibrin; protease-activated receptor; thromboelastography.

PubMed Disclaimer

Conflict of interest statement

Declaration of competing interests There are no competing interests to disclose.

Figures

Figure 1:

Role of platelets and platelet contraction in TEG. Citrated blood samples were collected by retroorbital bleed (RO) from wild-type (WT) mice (n=8) or WT mice depleted of circulating platelets (WT Plt-depleted) by injection of an anti-GPIbα antibody (R300, 1 mg/kg, n=4). For inhibition of platelet-mediated contraction, WT samples were treated with DMSO (n=4) or cytochalasin D (5 μg/ml, n=4) for 10 mins prior to TEG assay. Samples were mixed with CaCl2 and kaolin in plastic TEG cup and run immediately in a TEG 5000 analyzer and recorded for 1 hour. (A-C) TEG parameters: R time (A), α-angle (B) and MA (C). (D) Representative TEG traces. Data shown as mean ± SD. Statistical significance was determined by unpaired Student’s t-test (A) or one-way ANOVA with Tukey’s multiple comparison test. Symbols directly over bars represent significance compared to WT control. *P < .05, ****P < .0001.

Figure 2:

Role of αIIbβ3 integrin activation and ligand binding in TEG. Blood samples were analyzed from WT mice (n=8) or mice with megakaryocyte/platelet-specific deletion of Talin1 (Tln1 mKO, n=5). αIIbβ3 ligand binding was inhibited by treating WT samples with anti-αIIbβ3 antibody (Leo.H4, 75 μg/ml, n=6) for 10 mins prior to TEG assay. (A-C) TEG parameters: R time (A), α-angle (B) and MA (C). (D) Representative TEG traces. Data shown as mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test. Symbols directly over bars represent significance compared to control. **P< .01, ***P<0.001, ****P < .0001.

Figure 3:

Role of Rap1 GTPase signaling in TEG. Blood samples were analyzed from WT mice (n=8), mice with global deficiency in CalDAG-GEFI and P2Y12 (_Cdg1_−/− × _P2ry12_−/−, n=4) or mice with megakaryocyte/platelet-specific deletion of Rap1b alone (Rap1b mKO, n=4) or both Rap1a and Rap1b (Rap1a/b mKO, n=5). (A-C) TEG parameters: R time (A), α-angle (B) and MA (C). (D) Representative TEG traces. Data shown as mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test. Symbols directly over bars represent significance compared to control. **P< .01, ***P<0.001, ****P < .0001.

Figure 4:

Role of platelet PAR4 and GPVI in TEG. Blood samples were analyzed from WT mice (n=8), mice with megakaryocyte/platelet-specific deletion of PAR4 (PAR4 mKO, n=5), WT mice treated with anti-GPVI antibody to deplete GPVI on circulating platelets (JAQ1, 50 μg/mouse, n=4), or JAQ1-treated PAR4 mKO mice (n=3). (A-C) TEG parameters: R time (A), α-angle (B) and MA (C). (D) Representative TEG traces. Data shown as mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test. Symbols directly over bars represent significance compared to control. **P< .01, ****P < .0001.

Figure 5:

Role of Syk tyrosine kinase signaling in TEG. Blood samples were analyzed from WT mice (n=8), or WT (n=5) or PAR4 mKO (n=4) mice with addition of the Syk inhibitor PRT-2607 (20 μM) 10 mins prior to TEG assay. (A-C) TEG parameters: R time (A), α-angle (B) and MA (C). (D) Representative TEG traces. Data shown as mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test (A,C) or Kruskal-Wallis test with Dunn’s multiple comparison (B). Symbols directly over bars represent significance compared to control. **P < .01, ****P < .0001.

Figure 6:

Role of PAR1/PAR4 and Syk in human blood TEG. Healthy volunteer blood samples were analyzed with addition of DMSO (n=4), vorapaxar (Vora, 5 μM, n=3), BMS-986120 (BMS, 10 μM, n=3) and PRT-2607 (PRT, 20 μM, n=4) to inhibit PAR1, PAR4 and Syk, respectively, 10 mins prior to TEG assay. (A-C) TEG parameters: R time (A), α-angle (B) and MA (C). (D) Representative TEG traces. Data shown as mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test. Symbols directly over bars represent significance compared to control. *P < .05, **P< .01, *** P<0.001, ****P < .0001.

Figure 7:

PAR1/4 and GPVI play redundant roles in platelet activation and contraction in TEG. (1) Analysis of clot formation speed and clot strength in TEG is performed by recalcifying citrated whole blood and activating with kaolin to initiate coagulation. (2) Within several minutes, thrombin generation initiates fibrinogen cleavage to fibrin, which polymerizes and crosslinks. Additionally, thrombin activates platelets through protease activated receptors (PARs) (PAR4 on mouse platelets, PAR1 and PAR4 on human platelets). Both fibrin polymerization and platelet activation contribute to the speed of clot formation (α-angle). (3) As more fibrin is generated, platelets can also bind fibrin via GPVI for additional activation signaling, resulting in robust αIIbβ3 integrin activation and platelet-mediated contraction of the fibrin clot. Clot strength (MA) is entirely dependent on platelet contraction. (4) The end result is a tightly contracted whole blood clot with contracting platelets bound to fibrin. While loss of either platelet PARs or GPVI alone has limited impact on TEG parameters, loss of both leads to TEG traces similar to platelet-depleted blood samples. Created with

BioRender.com

References

1. Erdoes G, Koster A, Levy JH. Viscoelastic Coagulation Testing: Use and Current Limitations in Perioperative Decision-making. Anesthesiology; 2021; 135: 342–9. -PubMed
1. Raval JS, Burnett AE, Rollins-Raval MA, Griggs JR, Rosenbaum L, Nielsen ND, Harkins MS. Viscoelastic testing in COVID-19: a possible screening tool for severe disease? Transfusion (Paris); 2020; 60: 1131–2. -PMC -PubMed
1. Schmidt AE, Israel AK, Refaai MA. The Utility of Thromboelastography to Guide Blood Product Transfusion. Am J Clin Pathol; 2019; 152: 407–22. -PubMed
1. Wikkelsø A, Wetterslev J, Møller AM, Afshari A. Thromboelastography (TEG) or thromboelastometry (ROTEM) to monitor haemostatic treatment versus usual care in adults or children with bleeding. Cochrane Database of Systematic Reviews; 2016; 2016. -PMC -PubMed
1. Serraino GF, Murphy GJ. Routine use of viscoelastic blood tests for diagnosis and treatment of coagulopathic bleeding in cardiac surgery: Updated systematic review and meta-analysis. Br J Anaesth; 2017; 118: 823–33. -PubMed

Protease-activated receptors and glycoprotein VI cooperatively drive the platelet component in thromboelastography - PubMed (original) (raw)