Blood coagulation is a host defense mechanism that maintains the closed circulatory system when blood vessel integrity is compromised. Pathological processes such as inflammation and atherosclerosis are associated with thrombosis. Thrombosis is also a major complication in gastrointestinal adenocarcinoma, particularly pancreatic carcinoma (39
). In addition to BSDL, pancreatic carcinoma cells express high levels of an oncofetal variant of the enzyme, the fetoacinar pancreatic protein (FAPP) (40
), missing 10 of 16 proline-rich repeats at the C-terminal end of the protein but containing the V3-like loop region (42
). This protein is found at elevated levels in most patients with pancreatic cancer (40
). We thus sought to define the role of circulating BSDL.
In this study we demonstrate that BSDL plays a physiological role in thrombus formation, a unique function for a lipase. Our in vitro results indicate that BSDL, by increasing the amount of activated αIIbβ3, enhances both the aggregation and the spreading of thrombin-activated platelets. The enzyme also induces calcium mobilization, a marker of platelet activation. The V3-like loop structure of BSDL mimics these effects through interaction with the chemokine receptor CXCR4. In vivo, we show that BSDL accumulates in a CXCR4-dependent interaction on platelets at the site of laser-induced injury, enhancing platelet accumulation. These results indicate that BSDL acts as a chemokine to optimize platelet aggregation and platelet thrombus formation.
There have been several studies on the effects of chemokines on platelets in vitro (19
). Based on the action of SDF-1 on platelets, some chemokines have been defined as weak platelet agonists (25
) because of the increased platelet aggregation induced by thrombin or ADP after chemokine stimulation. Furthermore Clemetson et al. (19
) have shown that SDF-1 induces intracellular calcium mobilization in platelets. These effects are prevented when the production of thromboxane A2
is inhibited by aspirin. In the present study, we observed that BSDL or a peptide with sequence homology to its V3-like loop induces calcium mobilization and increases platelet aggregation and αIIb
activation in thrombin-activated platelets. These effects were prevented using either blocking antibodies or a specific inhibitor (AMD3100) against CXCR4 or aspirin to prevent the generation of thomboxane A2
. Based on our results and those described for chemokines (25
), we conclude that BSDL acts as a chemokine on platelets.
Two studies have previously determined the concentration of circulating BSDL in serum at 1.5 ± 0.5 μg/l (9
). We have previously shown that BSDL is found in the circulation both free and bound to apolipoprotein B–containing lipoproteins (7
). Further analysis of the original data allowed us to calculate the distribution of free BSDL versus BSDL bound to apolipoprotein B–containing lipoproteins bound BSD. We determined that 35% ± 7% of the total circulating BSDL was found free in plasma versus 57% ± 7% bound to apolipoprotein B–containing lipoproteins (VLDL, LDL, and chylomicrons). However, we present here evidence that BSDL is also detectable in platelets and released into the blood circulation upon activation. It is then possible that the quantity of local BSDL increases greatly during the formation of a platelet thrombus. This explains why we can detect endogenous BSDL at sites of laser-induced injury (Figure ). Therefore, we conclude that in vivo, the local concentration of BSDL at the sites of injury is much higher than the concentration of circulating BSDL in blood. Although some BSDL is associated with platelets within the thrombus, some also concentrates on the vessel wall. Since in vitro studies have shown that BSDL can interact with activated endothelial cells to promote wound healing, we determined whether BSDL could modify the prostacyclin and nitric oxide production of HUVECs in vitro. Our results showed that indeed BSDL increases the production of prostacyclin by thrombin-treated HUVECs. This may explain why in vivo a fraction of detected BSDL was interacting with (activated) endothelium at the site of the injured vessel (see Figure D).
BSDL may play a role in cancer-associated thrombosis. Even if a direct link between an increase in circulating BSDL (or BSDL stored in platelets) and a prothrombotic tendency is to date not known, pancreatic carcinoma is associated with a prothrombotic state (43
). Furthermore, our laboratory has identified the protein FAPP as an oncofetal isoform of BSDL (40
). Last, the concentration of circulating BSDL (including FAPP) is enhanced during the development of a pancreatic cancer (41
). Together these results may identify BSDL and FAPP as important agents linking thrombosis to pancreatic carcinoma. These results may also suggest a role for the V3 loop of gp120 in thrombosis associated with HIV-1 infection (26
). Further, we conclude that BSDL interaction with CXCR4 may be a new target for antithrombotic therapy in these and other disease states.