Soluble fibrin causes an acquired platelet glycoprotein VI signaling defect: implications for coagulopathy - PubMed (original) (raw)

. 2017 Dec;15(12):2396-2407.

doi: 10.1111/jth.13863. Epub 2017 Oct 27.

Affiliations

Soluble fibrin causes an acquired platelet glycoprotein VI signaling defect: implications for coagulopathy

M Y Lee et al. J Thromb Haemost. 2017 Dec.

Abstract

Essentials Collagen and thrombin when used simultaneously generate highly activated platelets. The effect of thrombin stimulation on subsequent glycoprotein VI (GPVI) function was observed. Soluble fibrin, but not protease activated receptor (PAR) activation, prevented GPVI activation. Circulating soluble fibrin in coagulopathic blood may cause an acquired GPVI signaling defect.

Summary: Background In coagulopathic blood, circulating thrombin may drive platelet dysfunction. Methods/Results Using calcium dye-loaded platelets, the effect of thrombin exposure and soluble fibrin generation on subsequent platelet GPVI function was investigated. Exposure of apixaban-treated platelet-rich plasma (12% PRP) to thrombin (1-10 nm), but not ADP or thromboxane mimetic U46619 exposure, dramatically blocked subsequent GPVI activation by convulxin, collagen-related peptide or fibrillar collagen. Consistent with soluble fibrin multimerizing and binding GPVI, the onset of convulxin insensitivity required 200-500 s of thrombin exposure, was not mimicked by exposure to PAR-1/4 activating peptides, was not observed with washed platelets, and was blocked by fibrin polymerization inhibitor (GPRP) or factor XIIIa inhibitor (T101). PAR-1 signaling through Gαq was not required because vorapaxar blocked thrombin-induced calcium mobilization but had no effect on the ability of thrombin to impair GPVI-signaling. Convulxin insensitivity was unaffected by the metalloprotease inhibitor GM6001 or the αIIb β3 antagonist GR144053, indicating negligible roles for GPVI shedding or αIIb β3 binding of fibrin. Thrombin treatment of washed platelets resuspended in purified fibrinogen also produced convulxin insensitivity that was prevented by GPRP. Exposure of apixaban/PPACK-treated whole blood to thrombin-treated fibrinogen resulted in > 50% decrease in platelet deposition in a collagen microfluidic assay that required soluble fibrin assembly. Conclusions Conversion of only 1% plasma fibrinogen in coagulopathic blood would generate 90 nm soluble fibrin, far exceeding ~1 nmGPVI in blood. Soluble fibrin, rather than thrombin-induced platelet activation throuh PAR-1 and PAR-4, downregulated GPVI-signaling in response to stimuli, and may lead to subsequent hypofunction of endogenous or transfused platelets.

Keywords: GPVI; coagulopathy; fibrin; platelet; thrombin; trauma.

© 2017 International Society on Thrombosis and Haemostasis.

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Conflict of interest statement

Conflict-of-interest disclosure: No conflicts to disclose.

Figures

Figure 1

Figure 1. Thrombin but not ADP or U46619 blocks subsequent platelet GPVI activation by convulxin

(A) Platelet activation by thrombin for 480 seconds causes a significant reduction in subsequent convulxin-induced calcium response. This effect was apparent for doses of thrombin (1 – 10 nM) treatment of diluted (12%), apixaban-treated PRP. (B) Platelet activation by ADP does not significantly attenuate subsequent convulxin-induced calcium response. (C) Platelet activation by the thromboxane analog, U46619, did not significantly attenuate subsequent convulxin-induced calcium response. (C, convulxin; T, thrombin; A, ADP; U, U46619).

Figure 2

Figure 2. Thrombin treatment of platelets blocks subsequent activation via fibrillar collagen when measuring calcium mobilization

Various doses of thrombin (A: 10 nM; B: 5 nM; C: 2 nM) prevent further downstream platelet activation via collagen (Chrono-log; 10 µg/mL), fully consistent with the previous findings using convulxin. Additionally, collagen and convulxin show significantly different kinetic profiles of GPVI signaling which can be attributed to the molecular composition of each species. Collagen, a larger and more fibrillar molecule, activates GPVI in a slower but more sustained manner, while convulxin elicits a rapid and transient response.

Figure 3

Figure 3. Effect of thrombin dose and exposure time to drive convulxin-insensitivity

(A) When platelets were treated with low dose thrombin (1 nM), the reduction of convulxin-sensitivity was time-dependent with strong onset detected between 335 and 480 sec of thrombin incubation. (B) When platelets were activated by a slightly higher thrombin dose (2 nM), thrombin-induced convulxin-insensitivity was detected after 200 sec of thrombin incubation. (C) Sensitivity to convulxin decreased with thrombin incubation time (data fitted with a Hill function) with more rapid onset observed at higher thrombin dose. (C, convulxin; T, thrombin).

Figure 4

Figure 4. Attenuation of convulxin sensitivity was not observed by pretreatment with PAR-1 and PAR-4 agonist peptides

(A) Despite calcium mobilization through PAR-1 and PAR-4, platelet activation by PAR-1 and PAR-4 specific agonist peptides (SFLLRN and AYPGKF) did not reduce subsequent convulxin-insensitivity, as was observed with thrombin. (B) Vorapaxar, a PAR-1 antagonist, blocked thrombin-induced calcium signaling. (C) The convulxin-insensitivity after thrombin treatment was still apparent in the presence of vorapaxar. (PAR-1=SFLLRN activation peptide; PAR-4=AYPGKF activation peptide; V, vorapaxar; T, thrombin; C, convulxin).

Figure 5

Figure 5. Inhibition of fibrin polymerization with GPRP prevents thrombin-induced convulxin-insensitivity

(A) The thrombin-induced attenuation of platelet calcium GPVI signaling was not observed when GPRP was present. (B) Because platelet activation by ADP does not cause fibrinogen to polymerize into fibrin, GPRP has no effect on subsequent convulxin response. (C) Following thrombin stimulation of platelets, fibrin results in a sustained calcium mobilization that returned to unstimulated levels in the presence of GPRP.

Figure 6

Figure 6. Inhibition of cross-linking enzyme FXIIIa with T101 results in significant restoration of platelet GPVI activity and inhibition of ADAM10 shows no effect of GPVI shedding

(A) Calcium dye-loaded platelets were incubated with 1 mM T101 for 10 minutes prior to activation with thrombin (2–20 nM). In the presence of the FXIIIa inhibitor, fibrin still polymerizes but cannot cross-link to form the traditional mesh network which is crucial for clot stabilization. The plot shows the effect of T101 towards greatly restoring convulxin-induced platelet activation. (B) Platelet GPVI exhibits a marked increase in sensitivity, especially at intermediate concentrations of thrombin (n=4 donors, * p<0.05). (C) Thrombin-induced convulxin-insensitivity does not require GPVI shedding. Activation of platelets by thrombin resulted in attenuation of subsequent convulxin-induced calcium mobilization even in the presence of GM6001, which blocks GPVI shedding. When GPRP was present to block fibrin polymerization, thrombin treatment had no effect convulxin sensitivity, even in the presence of GM6001 which blocks GPVI shedding.

Figure 7

Figure 7. Thrombin activation of washed platelets in purified fibrinogen reduced subsequent activation by convulxin, an effect blocked by GPRP

(A) Schematic of experimental protocol. (B) In a washed platelet and fibrinogen (500 nM) mixture, low dose of thrombin (1 nM) at 480 seconds incubation time significantly attenuated subsequent convulxin response.

Figure 8

Figure 8. Under thrombin-free conditions, presence of soluble fibrin in whole blood reduces platelet adhesion on collagen under flow

(A) Schematic of experimental protocol. Fluorescent fibrinogen was exposed to 2.5 nM thrombin with either 5 mM GPRP (inhibit fibrin formation) or HBS (control). After 300 s, the thrombin was quenched with 100 µM PPACK. This reacted fibrinogen/fibrin solution was diluted by a factor of 10 into PPACK/apixaban-inhibited whole blood with either 5 mM GPRP or HBS and then perfused through an 8-channel microfluidic device at 200 s−1 over a collagen surface. (B) GPRP increased platelet deposition on the collagen surface after 60 s. (C) Fibrin co-deposition with platelets was significantly decreased with 5 mM GPRP. (D) At 120 s, soluble fibrin monomer (GPRP present) resulted in more platelet deposition to collagen, while soluble fibrin polymer (no GPRP) resulted in less platelet adhesion to collagen. (E) At 240 s, platelet deposition and aggregation on collagen was quite pronounced in the presence of GPRP which blocked fibrin co-deposition, as expected.

Comment in

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