Enhanced procoagulant activity of platelets after chemotherapy in non-small cell lung cancer

Abstract

The procoagulant status of patients with non-small cell lung cancer (NSCLC) after chemotherapy is poorly characterized and the role of platelets in hypercoagulative state of NSCLC is unknown. The aim of this study was to evaluate the procoagulant activity (PCA) of platelets in NSCLC before and after chemotherapy. The subjects were 52 patients newly diagnosed with NSCLC. The patients had decreased clotting time compared with healthy subjects, and the thrombin-antithrombin complex increased 2.5-fold after chemotherapy. Platelets in the patients after chemotherapy had enhanced phosphatidylserine (PS) exposure, and shortened coagulation time as well as increased thrombin and fibrin formation of platelets compared with those before chemotherapy. Platelet-derived microparticles increased 2-fold at day 1 and peaked at day 2 post-chemotherapy. Treatment of cisplatin in vitro also resulted in upregulated intrinsic FXa and thrombin formation on platelets with a dose-dependent manner. Platelets treated with aspirin significantly decreased PCA. However, lactadherin blocked PS and inhibited the PCA approximately by 70%. Seven days after chemotherapy, PCA of platelets restored to the baseline as that before chemotherapy, indicating that within a week of chemotherapy patient platelets are highly procoagulant and effective intervention should be taken in case of thrombosis. Our results suggested that platelets after chemotherapy had elevated PCA and may contribute to the hypercoagulative state of NSCLC. Prophylactic anti-coagulant combined with anti-platelet therapy may play an inhibitory role in thrombotic complications in NSCLC.

Keywords: Chemotherapy; non-small cell lung cancer; platelet; procoagulant activity; thrombosis.

Figure 1.

Clotting time and TAT in patients with NSCLC within 7 d after chemotherapy. Blood of patients before and after chemotherapy was isolated, and clotting time of whole blood (a) and TAT level in serum (b) were measured. Data are present as mean ± SD, *P < 0.05 vs day 0, **P < 0.01 vs day 0.

Figure 2.

PS expression and PCA of platelets after chemotherapy. Platelets were isolated from patients before and after chemotherapy. (a) PS and TF expression on platelets were measured respectively with Alexa 488-lactadherin (green) and Alexa 647-anti CD142 (red) by flow cytometry. (b) Coagulation time was measured by coagulometer and thrombin and fibrin formation were evaluated with microplate reader. Data are present as mean ± SD, *P < 0.05 vs day 0, **P < 0.01 vs day 0.

Figure 3.

The number of PMPs after chemotherapy. Platelets were isolated from patients before and after chemotherapy. (a,b) The number of total MPs and PMPs were analyzed by flow cytometry. PMPs were characterized by CD41a expression. Data are present as mean ± SD, **P < 0.01 vs day 0. (c) Confocal microscopy images showed quiescent platelets (left, arrow) and apoptotic platelets positive for lactadherin staining (right, arrow) with MPs (right, arrowheads) at day 1. Bars represent 2 µm.

Figure 4.

The effect of cisplatin on PCA of platelets. Platelets were isolated from healthy subjects and treated with cisplatin of different concentrations for 1 h in vitro. Coagulation time (a), intrinsic/extrinsic FXa and thrombin (b) as well as fibrin (c) formation were evaluated. Data are present as mean ± SD, *P < 0.05 vs untreated, **P < 0.01 vs untreated. (c) After 1µM cisplatin treatment of 1 h, quiescent platelets (left), activated platelets (middle) and apoptotic platelets with MPs (right) were shown by scanning microscopy images. Bars represent 1 µm.

Figure 5.

The effect of anti-platelet agents and lactadherin on the PCA. After 1 µM cisplatin treatment of 1 h, platelets were treated with aspirin (100 nM) and lactadherin (2 nM). Coagulation time (a), intrinsic/extrinsic FXa and thrombin (b) as well as fibrin (c) formation of platelets were measured. Data are present as mean ± SD, *P < 0.05 vs control, **P < 0.01 vs control.