Platelet protein S limits venous but not arterial thrombosis propensity by controlling coagulation in the thrombus

Abstract

Anticoagulant protein S (PS) in platelets (PSplt) resembles plasma PS and is released on platelet activation, but its role in thrombosis has not been elucidated. Here we report that inactivation of PSplt expression using the Platelet factor 4 (Pf4)-Cre transgene (Pros1lox/loxPf4-Cre+) in mice promotes thrombus propensity in the vena cava, where shear rates are low, but not in the carotid artery, where shear rates are high. At a low shear rate, PSplt functions as a cofactor for both activated protein C and tissue factor pathway inhibitor, thereby limiting factor X activation and thrombin generation within the growing thrombus and ensuring that highly activated platelets and fibrin remain localized at the injury site. In the presence of high thrombin concentrations, clots from Pros1lox/loxPf4-Cre- mice contract, but not clots from Pros1lox/loxPf4-Cre+ mice, because of highly dense fibrin networks. Thus, PSplt controls platelet activation as well as coagulation in thrombi in large veins, but not in large arteries.

Graphical abstract

Figure 1.

Generation of a mouse model lacking PS expression in platelets (Pros1^lox/loxPf4-Cre⁺). (A) Genotyping results of mice with normal PS expression (Pros1^lox/loxPf4-Cre⁻), Pros1^lox/−, and Pros1^lox/loxPf4-Cre⁺ mice using genomic DNA extracted from ear tissues. The presence of lox sequences in Pros1 alleles and of Pf4-Cre transgene was evaluated by 2 independent multiplex PCRs. PCR products were then subjected to electrophoresis. The lox band had a lower molecular weight (338 bp) compared with the null band (570 bp), in accordance with Saller et al; the Pf4-Cre⁺ band has a higher molecular weight (450 bp) in comparison with the internal PCR control (324 bp). (B) PS antigen levels in platelet lysates from Pros1^lox/loxPf4-Cre⁻, Pros1^lox/loxPf4-Cre⁺, and Pros1^lox/− mice. Results are expressed as mean ± standard error of the mean (SEM) of percentage relative to the pooled normal platelet lysate. **P ≤ .01; ****P ≤ .0001. (C) PS antigen levels in plasma samples from Pros1^lox/loxPf4-Cre⁻, Pros1^lox/loxPf4-Cre⁺, and Pros1^lox/− mice. Results are expressed as mean ± SEM of percentage relative to the pooled normal plasma. ns, not significant; ****P ≤ .0001. (D-E) PS isoforms in plasma and platelet after activation by thrombin. Western blotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions was performed using a monoclonal antibody raised against murine PS (D) and a polyclonal antibody against human PS (E). P, platelet; S, platelet releasate; Thr, thrombin. (F) Confocal microscopy of resting and thrombin-activated mouse Pros1^lox/loxPf4-Cre⁻ and Pros1^lox/loxPf4-Cre⁺ platelets, stained for -tubulin (in magenta), CD62p (in green), and PS (in red). Scale bar, 2 μm. Acquired z stacks were used for volumes and surface rendering by Imaris software.

Figure 2.

Lack of PS in platelets does not affect platelet activation state in steady state, platelet aggregation ex vivo, and platelet-dependent VTE in vivo. (A) PF4 antigen levels in plasma from Pros1^lox/loxPf4-Cre⁻ and Pros1^lox/loxPf4-Cre⁺ mice (ns, not significant; P = .145). Data are expressed as median. The normal range of the PF4 antigen concentration in mouse plasma (0.26-4.70 µg/mL) provided by the manufacturer is indicated in the y-axis by dashed lines. (B) Flow cytometry analysis of CD62p and CD41 on resting and convulxin (10 µg/mL)-activated or thrombin (10 U/mL)-activated platelets. (C) Platelet Function Analyzer-100 closure time using collagen-ADP and collagen-epinephrine (collagen-Epi) cartridges to activate platelets in whole blood from Pros1^lox/loxPf4-Cre⁻ and Pros1^lox/loxPf4-Cre⁺ (n = 5/genotype). Data are expressed as mean ± SEM. (D) Aggregation of washed platelets suspensions were initiated by adding different concentrations of collagen, ADP (in presence of 2 mg/mL fibrinogen), and thrombin. Results are given as mean ± SEM of at least 3 independent experiments; each of them was obtained from platelet pools (n = 4 mice/genotype). (E) Venous thromboembolism model induced by an intravenous injection of collagen-epinephrine in Pros1^lox/loxPf4Cre⁻ (straight line; n = 16) and mice Pros1^lox/loxPf4Cre⁺ (dashed line; n = 13) mice.

Figure 3.

Lack of PS in platelets affects global hemostasis assessment ex vivo and in vivo. (A-B) Whole blood activation of coagulation and clot polymerization determined by ROTEM analysis rotational (thromboelastography). (A) Extrinsic‐activated rotational thromboelastometry (EXTEM) assay, (B) extrinsically activated thromboelastometric test with cytochalasin D (FIBTEM). CT, coagulation time; CFT, clot formation time; α, α-angle; CFR, clot formation rate, MCF, maximum clot firmness; MCE, maximum clot elasticity. (C-D) Total thrombus-formation analysis on whole blood on collagen and TF-coated chip (AR-chip) at low (240 s⁻¹, C) and high (600 s⁻¹, D) shear rate. (E-F) Bleeding time (E) and blood loss (F) were measured after 2-mm tail transection in Pros1^lox/loxPf4-Cre⁻ (n = 7), Pros1^lox/loxPf4-Cre⁺ (n = 7), and Pros1^lox/− (n = 5) mice. All data are expressed as mean ± SEM. ns, not significant; *P < .05; **P ≤ .01; ***P ≤ .001.

Figure 4.

Platelets PS limits thrombus propensity and affects thrombus composition. (A-C) TF-induced venous thromboembolism using high dose of recombinant TF in A (1/2 dilution of Innovin; ∼4.3 nM TF) and low dose in panels B and C (1/8 dilution of Innovin; ∼1.1 nM TF). In panel A, no differences were found between Pros1^lox/loxPf4-Cre⁻ (straight line; n = 14) and Pros1^lox/loxPf4-Cre⁺ (dashed line, n = 14). (B) Pros1^lox/loxPf4-Cre⁺ (dashed line; n = 14) and Pros1^lox/− (dotted line, n = 14) mice showed higher mortality than Pros1^lox/loxPf4-Cre⁻ (straight line; n = 14) mice. (C) Thrombus formation in FeCl₃-injured mesenteric arterioles recorded by intravital microscopy in Pros1^lox/loxPf4-Cre⁻ and Pros1^lox/loxPf4-Cre⁺ mice, representative experiment (n = 7/genotype). Recorded occlusion time is shown in panel D. (E-G) Thrombi were collected 20 minutes after FeCl₃ challenge and processed to confocal microscopy, pictures were taken close to the lesion side and on the top of the thrombus as shown in panel E. Confocal microscopy of the thrombi analyzed 20 minutes after the FeCl₃ injury and stained for FXa and thrombin and CD41 (F; scale bar, 50 μM) as well as for insoluble fibrin, CD41, and CD62p (G; scale bar, 50 μM). Data are expressed as mean ± SEM. *P < .05; **P ≤ .01; ***P ≤ .001.

Figure 5.

Platelets PS limits thrombosis propensity in the vena cava, but not in the carotid artery. (A) Evaluation of the thrombus volume in the vena cava 30 minutes after the FeCl₃ injury, using ultra-high-frequency US (n = 4/genotype). Using the Vevo 3100 imaging system (Fujifilm VisualSonics Canada) and a MX550S linear array transducer (bandwidth, 32-55 MHz; center frequency, 40 MHz), 3-dimensional image series were acquired after 30 minutes of thrombus induction at 76-µm intervals. Using the VevoLab software package (Fujifilm VisualSonics), the 3-dimensional image series were used to make a 3-dimensional reconstruction of the inferior vena cava or carotid artery and a 3-dimensional reconstruction of the thrombus. The vessel and the thrombus area were measured by the software in cubic millimeters. The thrombus volume (%) refers to the volume (in cubic millimeters) that the thrombus occupies inside the volume (mm³) of the vessel. (B) Thrombi collected at the end of the experiment (=30 minutes after the FeCl₃ injury) and processed for immunohistochemistry; the sections were performed in the middle of the vena cava thrombus, as shown. Hematoxylin and eosin staining (HE) and immunohistochemistry for insoluble fibrin, PS, and CD31 were realized in Pros1^lox/loxPf4Cre⁻ and Pros1^lox/loxPf4Cre⁺ vena cava thrombi 30 minutes after the FeCl₃ injury. Scale bar, 500 µm. (C) Evaluation of the thrombus volume in the carotid artery 30 minutes after the FeCl₃ injury, using ultrahigh-frequency US (n = 3/genotype). For the method of evaluation of the thrombus volume, see panel A. (D) HE and immunohistochemistry for insoluble fibrin, PS, and CD31 were realized in Pros1^lox/loxPf4Cre⁻ and Pros1^lox/loxPf4Cre⁺ carotid artery thrombi 30 minutes after the FeCl₃ injury. Scale bar, 200 µm.

Figure 6.

Effect of the lack of PS in platelets on clot contraction. (A) Representative images of clot contraction at different points after induction with thrombin 10 U/mL in Pros1^lox/loxPf4Cre⁻ and Pros1^lox/loxPf4Cre⁺ PRP (n = 3/genotype). (B) Representative SEM images of the contracted clot at the external surface at different magnifications: scale bar, 10, 2, and 1 µm for ×3200, ×10 000, and ×30 000, respectively). (C) Representative SEM images of the contracted clot in the middle of the clot at different magnifications: scale bar, 2 and 1 µm for ×10 000 and ×30 000, respectively; scale bar, 500 nm for ×50 000. (D) Quantification of fibrin network density in Pros1^lox/loxPf4Cre⁻ and Pros1^lox/loxPf4Cre⁺ mice. Measurements are presented as mean ± SEM. **P ≤ .01.

Figure 7.

Absence of PS expression in platelets affects resistance to APC- and TFPI-dependent PS activity. (A-B) APC-dependent activity assessed by thrombin generation in PRP (150 × 10⁹/L platelets). Representative thrombin generation curves in presence of different amount of WT mAPC and L38D mAPC or buffer (no APC) in PRP from Pros1^lox/loxPf4-Cre⁻ (A) and Pros1^lox/loxPf4-Cre⁺ mice (B). (C-D) Washed platelet suspension (150 G/L) from Pros1^lox/loxPf4-Cre⁻ (C) and Pros1^lox/loxPf4-Cre⁺ mice (D) were reconstituted in platelet-free plasma (PFP) from F8^−/−Pros1^−/− mice, lacking plasma PS to evaluate the contribution of platelet PS to APC activity. (E-F) To evaluate the contribution of platelet PS to the thrombin potential, thrombin generation was measured in Pros1^lox/loxPf4-Cre⁻ and Pros1^lox/loxPf4-Cre⁺ mice. ETP values from PRP (200 G/L platelets) (E) and PFP (F) are shown for both genotypes. (G) ETP change between PRP and PFP [(ETP_PRPx100/ETP_PFP)-100)] in Pros1^lox/loxPf4-Cre⁻ and Pros1^lox/loxPf4-Cre⁺ mice. Data are expressed as mean ± SEM. *P < .05; **P ≤ .01. (H) FXa in activated Pros1^lox/loxPf4-Cre⁻ and Pros1^lox/loxPf4-Cre⁺ platelets; representative experiment obtained from a pool of 5 to 6 mice/genotype.