Subendothelium Research Articles

Antibodies to CD36 (GPIV) Inhibit Platelet Adhesion to Subendothelial Surfaces Under Flow Conditions

The membrane glycoprotein CD36 (glycoprotein [GP] IV) has previously been shown to accelerate the initial interaction of platelets with purified type I collagen in both static and flow systems. In the present study, the role of CD36 on platelet interaction with physiologically relevant collagenous surfaces was addressed. Using arterial subendothelium (SE) and endothelial cell extracellular matrix (ECM), studies were performed under flow conditions with annular and parallel-plate perfusion chambers, respectively, at a shear rate of 800 s-1 for 2, 5, and 10 minutes. Perfusates consisted of citrated normal blood samples incubated with Fab fragments of a monospecific polyclonal anti-CD36 antibody or with each of three new anti-CD36 monoclonal antibodies (MoAbs) that inhibit platelet adhesion to purified type I collagen in a static system (131.4, 131.5, and 131.7). Perfusions over SE were also carried out using citrated blood samples from a Naka-negative donor, whose platelets lack CD36. Morphometric evaluation of the perfused samples showed that polyclonal anti-CD36 Fab and the three monoclonal anti-CD36 antibodies inhibited platelet adhesion to the two substrates by 40% after 2 minutes of perfusion and by 30% after 5 minutes (P < .005 on SE and P < .01 on ECM), but at 10 minutes, significant inhibition was seen only on SE with polyclonal anti-CD36 Fab. Similar inhibitions were seen with Naka-negative platelets on SE. These studies demonstrate that CD36 plays a role in the early stages of platelet adhesion to physiologically relevant subendothelial surfaces.

Recombinant human erythropoietin treatment improves platelet function in uremic patients

The effect of recombinant human erythropoietin (rHuEPO) on primary hemostasis was tested in 19 hemodialyzed patients. Bleeding time, platelet aggregation and platelet interaction with vessel subendothelium (SE) under flow conditions were determined before treatment and after patients reached hematocrits greater than or equal to 30%. Two thrombotic events (an acute myocardial infarction and an AV fistula clotting) were recorded during the early stages of treatment. A shortening of average bleeding times (P less than 0.01), an increase in platelet count (P less than 0.01) and an improvement of platelet aggregation (P less than 0.01) and of platelet-SE interaction (P less than 0.01) were observed. A low correlation index was found between hematocrit and bleeding time (r = -0.351, P less than 0.05). To assess a possible effect of rHuEPO on platelet function, the same parameters were evaluated before and after receiving three doses of rHuEPO (40 U/kg i.v. post-hemodialysis) in 14 of the patients. No changes in platelet or erythrocyte counts were observed, the mean bleeding time remained unchanged, but platelet aggregation induced by arachidonic acid (P less than 0.05), ADP (P less than 0.01) and ristocetin (P less than 0.05) improved. Perfusion studies confirmed moderate but significant increases in the parameters that quantify platelet-SE interaction (P less than 0.05). Improvement of ADP-induced aggregation correlated with the increase of platelet adhesion to SE (r = 0.675, P less than 0.05). We conclude that rHuEPO treatment improves primary hemostasis in uremia through an increase of red cell mass but also through a beneficial effect on platelet function, which is independent of the hematocrit rise.

Plasma from systemic lupus erythematosus patients with antiphospholipid antibodies promotes platelet aggregation. Studies in a perfusion system.

The possible platelet-aggregating effect of plasma from systemic lupus erythematosus (SLE) patients (n = 19) was investigated under flow conditions. Aliquots of the SLE plasmas with (n = 10) or without (n = 9) anticardiolipin antibodies (ACAs) were added to anticoagulated blood (1:20, vol/vol). Plasma from normal donors was used as a control. Blood was incubated for 15 minutes at 37 degrees C and then perfused through annular chambers containing denuded arterial segments. Perfusions were performed for 10 minutes at a shear rate of 800 sec-1. The interaction of platelets with vessel subendothelium (SE) was morphometrically evaluated in thin sections. In control experiments, the percentage of the SE covered with platelets was 23.6 +/- 4.3% (mean +/- SD). Large aggregates (more than 5 microns in height) covering 11.8 +/- 5.7% of the exposed SE were noted. The deposition of platelets was statistically increased (38.5 +/- 7.6%, p less than 0.01 versus control) in the presence of SLE plasmas with demonstrated antiphospholipid antibodies (APAs). The formation of large aggregates was also augmented (30.3 +/- 5.9%, p less than 0.01 versus control). A similar response was obtained after addition of affinity-purified immunoglobulin G and immunoglobulin M fractions from two patients with ACAs. SLE plasmas with no detectable APAs did not influence the morphometric parameters studied. Results of the present study indicate that the presence of APAs in SLE plasma promotes platelet aggregation under flow conditions. These observations may help to explain the pathophysiology of the thrombotic events occurring in patients with APAs.

Von Willebrand Factor and platelet interactions with the vessel wall

von Willebrand factor (vWF) is a multimeric glycoprotein which has a dual role in haemostasis, functioning as carrier protein for Factor VIII and mediating platelet adhesion to exposed subendothelium (SE). vWF interacts with components of the SE such as collagen and heparin-like glycosaminoglycans as well as with two platelet membrane receptors: glycoprotein (GP) Ib and GPIIb/IIIa. These multiple binding functions explain its definition as an adhesive protein. vWF promotes platelet adhesion at the high shear rates which correspond to the rheologic conditions of the microcirculation or of narrowed arterial vessels. The role of the vWF-GPIb interaction in platelet adhesion is well known; that of the vWF-GPIIb/IIIa interaction has been more recently demonstrated through the use of monoclonal antibodies (MAbs) or synthetic peptides blocking vWF-binding to GPIIb/IIIa. In addition, perfusion studies in native, non-anticoagulated blood emphasize the concept that vWF is also essential for thrombus formation at high shear stress. Thus, vWF fragments, synthetic peptides or MAbs blocking the functional domains of vWF represent potential therapeutic strategies to prevent the development of thrombosis.

Comparison between Human Umbilical Artery and Rabbit Abdominal Aorta as Substrata for Platelet Adhesion and Platelet Thrombus Formation under Flow Conditions

Rabbit abdominal aortas and human umbilical arteries are currently used as substrata for the study of platelet adhesion and aggregate formation under flow conditions. Using immunohistochemical and ultrastructural methods, we have analyzed both vessel surfaces. The reactivity towards platelets of the subendothelium (SE) exposed on these vessels after mechanical or enzymatic digestion (alpha-chymotrypsin) was morphometrically quantified and the nature of the interaction studied in the electron microscope. After mechanical damage, the ultrastructural study of rabbit aortas showed a clearly defined internal elastic lamina (IEL). In contrast, umbilical vessels lacked a consistent IEL and masses of amorphous material often located deeper in the media were the main constitutents of the SE. Immunohistochemical labeling of the von Willebrand factor bound to both types of vessel differed considerably. Quantification of platelet interactions after perfusion of citrated blood showed qualitative differences between mechanically damaged rabbit or human vessels. Enzymatic digestion produced a more thrombogenic surface on rabbit aortas (p less than 0.01 vs. nondigested), but decreased their reactivity towards platelets on umbilical arteries (p less than 0.01 vs. nondigested). The ultrastructural study of the interacting platelets revealed that aggregates, when present, were found on the extracellular matrix underlying endothelial cells of rabbit aortas, but interacting with fibrillar structures probably derived from cell elements of the media in the case of umbilical arteries. These findings indicate that rabbit aortas and umbilical arteries possess structural characteristics that result in different thrombogenic properties with respect to circulating platelets.

Uremic platelets have a functional defect affecting the interaction of von Willebrand factor with glycoprotein IIb-IIIa

Uremic patients have an impaired platelet function that has been related to membrane glycoprotein (GP) abnormalities. Using a perfusion system, we have studied the interaction of normal and uremic platelets with vessel subendothelium (SE) under flow conditions. Reconstituted blood containing washed platelets, purified von Willebrand factor (vWF) (1 U/mL), and normal washed red blood cells was exposed to de- endothelialized rabbit segments for 10 minutes at two different shear rates (800 and 1,600 seconds-1). In some experiments a monoclonal antibody to the GPIIb-IIIa complex (EDU3) was added to the perfusates. With normal platelets, the percentage of the vessel covered by platelets (%CS) was 23.1% +/- 3.7% at 800 seconds-1 and 30% +/- 4.3% at 1,600 seconds-1. Platelets were observed in contact or forming monolayers on vessel SE. EDU3 inhibited the spreading of normal platelets. The %CS (11.1% +/- 3.3%) was statistically decreased (P less than .01) and most of the platelets were observed in contact with the vessel surface. These data indicate that, under flow conditions, the interaction of vWF with GPIIb-IIIa can support the spreading of normal platelets in the absence of exogenous fibrinogen. Under the same experimental conditions, the interaction of uremic platelets with SE was markedly impaired at both shear rates studied (P less than .01 v normal platelets). The presence of EDU3 did not modify the interaction of uremic platelets. These results confirm the impairment of the platelet adhesion observed in uremic patients. Furthermore, they indicate the presence of a functional defect in the interaction of vWF with GPIIb-IIIa. The fact that perfusions with normal and uremic platelets in the presence of an antibody to the GPIIb-IIIa complex did not show any differences gives indirect evidence on a functionally normal interaction vWF/GPIb in uremic patients.

Uremic Platelets Have a Functional Defect Affecting the Interaction of von Willebrand Factor With Glycoprotein Ilb-IIIa

Uremic patients have an impaired platelet function that has been related to membrane glycoprotein (GP) abnormalities. Using a perfusion system, we have studied the interaction of normal and uremic platelets with vessel subendothelium (SE) under flow conditions. Reconstituted blood containing washed platelets, purified von Willebrand factor (vWF) (1 U/mL), and normal washed red blood cells was exposed to de-endothelialized rabbit segments for 10 minutes at two different shear rates (800 and 1,600 seconds-1). In some experiments a monoclonal antibody to the GPIIb-IIIa complex (EDU3) was added to the perfusates. With normal platelets, the percentage of the vessel covered by platelets (%CS) was 23.1% +/- 3.7% at 800 seconds-1 and 30% +/- 4.3% at 1,600 seconds-1. Platelets were observed in contact or forming monolayers on vessel SE. EDU3 inhibited the spreading of normal platelets. The %CS (11.1% +/- 3.3%) was statistically decreased (P less than .01) and most of the platelets were observed in contact with the vessel surface. These data indicate that, under flow conditions, the interaction of vWF with GPIIb-IIIa can support the spreading of normal platelets in the absence of exogenous fibrinogen. Under the same experimental conditions, the interaction of uremic platelets with SE was markedly impaired at both shear rates studied (P less than .01 v normal platelets). The presence of EDU3 did not modify the interaction of uremic platelets. These results confirm the impairment of the platelet adhesion observed in uremic patients. Furthermore, they indicate the presence of a functional defect in the interaction of vWF with GPIIb-IIIa. The fact that perfusions with normal and uremic platelets in the presence of an antibody to the GPIIb-IIIa complex did not show any differences gives indirect evidence on a functionally normal interaction vWF/GPIb in uremic patients.

Dose- and shear rate-dependent effects of heparin on thrombogenesis induced by rabbit aorta subendothelium exposed to flowing human blood.

The effect of heparin on thrombogenesis induced by the subendothelium of rabbit aorta was investigated in 24 healthy volunteers after intravenous injection of different doses (0, 1000, 2500, and 5000 IU). By using an ex vivo perfusion chamber system, the interaction between flowing blood and exposed subendothelium was measured at low (50 s-1), intermediate (650 s-1), and high (2600 s-1) wall shear rates. The low shear rate simulated blood flow in venous, the intermediate shear rate in arterial, and the high shear rate in small or stenosed arterial vessels. Deposition of fibrin, platelets, and platelet thrombi on vascular subendothelium (SE) was quantified by morphometrical and immunological techniques. Fibrin deposition prevailed at low shear rates and was only minimal at high shear rates (30 +/- 1% vs. 1 +/- 0.4% coverage of SE with fibrin, means +/- SEM, p less than 0.001). In contrast, the interaction of platelets with SE was more intense at high compared to low shear rates, as indicated by higher platelet adhesion (54 +/- 5% vs. 4 +/- 1% coverage of SE with platelets, p less than 0.001) and platelet thrombus volumes (4.8 +/- 1.3 vs. 0.5 +/- 0.1 microns 3/microns 2, p less than 0.001). Fibrin deposition on SE was inhibited by heparin in a dose-dependent manner and was abolished after high doses. In addition, high doses of heparin reduced the height and volume of platelet thrombi at low and intermediate wall shear rates, but no effect was found at the high shear rate. Our data show that heparin inhibits the formation of both fibrin and platelet thrombi on vascular subendothelium. The lack of effect of heparin on platelet thrombus formation at high shear rates indicates that thrombin modulates the growth rate and/or stability of platelet thrombi at low and intermediate shear rates, whereas additional factors may control platelet thrombus growth and stability at high shear conditions.

Culture-produced subendothelium. II. Effect of plasma, F.VIIIR:WF and fibronectin on interaction of normal platelets with normal and von Willebrand porcine aortic subendothelium

Culture-produced normal and von Willebrand (vWd) porcine aortic subendothelium (SE) was prepared on glass and fibronectin (FN)-coated surfaces. Washed porcine platelets reacted extensively with normal SE (on glass) as single adherent non-spread and spread (5%) platelets, whereas vWd SE (on glass)-platelet interaction was decreased with no spreading. Normal porcine plasma increased normal SE-platelet interaction 3 to 5-fold and spreading 5-fold (20–30%); vWd SE-platelet interaction was increased 2 to 4-fold with no spreading. vWd porcine plasma did not affect normal or vWd SE-platelet interaction or spreading. Purified porcine F.VIIIR:WF (0.5-2 U/ml) increased both normal and vWd SE-platelet interaction 2 to 4-fold without increasing spreading. Purified human FN (50–200 μg/ml) did not increase normal or vWd SE-platelet interaction but increased spreading 5 to 7-fold (25–35%) with normal SE. F.VIIIR:WF (2 U/ml) plus FN (200 μg/ml) increased normal SE-platelet interaction 4 to 5-fold and spreading 8 to 9-fold (41%) with extensive SE-associated microaggregate formation; vWd SE-platelet interaction was increased about 6-fold with no spreading. Platelets reacted more extensively with normal and vWd SE prepared on FN-coated surfaces than SE on glass. Normal SE (on FN)-platelet interaction increased 6 to 7-fold and spreading 12-fold (60%); vWd SE (on FN)-platelet interaction increased about 110-fold with about 10% spreading. Plasma (normal and vWd), and F.VIIIR:WF did not significantly increase normal or vWd SE (on FN)-platelet interaction or spreading. Results suggest a possible role for FN as a spreading factor in SE-platelet interactions.

Culture-produced subendothelium. I. Platelet interaction and properties.

Culture-produced subendothelium (SE) has been prepared from cultured porcine aortic endothelial cells (ECs) by a rapid freeze-thaw, ice-shearing method. En face preparations of this in situ SE material are essentially free of intact or damaged cells and cell debris and consisted of an extensive meshwork of microfibrillar and amorphous material. Washed porcine platelets reacted extensively with this SE material and were associated with the SE as single adherent platelets, single spread platelets, and varying-sized platelet aggregates or 'microthrombi'. Platelet aggregates were associated only with the damaged or frayed edges of the SE, and the platelets had undergone extensive SE-induced contraction and degranulation, as indicated by transmission electron microscopy. Platelet-SE interaction was affected by pH, calcium, platelet concentration, rapid shaking and exposure time. Platelet-SE interaction was significantly enhanced by the addition of 0.1-1% citrated plasma or purified porcine F.VIIIR:WF. Pretreatment of the SE with thrombin, elastase, neuraminidase or hyaluronidase had no effect on platelet-SE interaction, whereas pretreatment with pepsin, plasmin, trypsin, alpha-chymotrypsin or collagenase decreased or completely abolished all platelet-SE interaction. Extraction of the SE with various solutions (high salt, detergents, etc.) had no effect on platelet-SE interaction, only solutions containing sodium dodecyl sulfate completely abolished all platelet-SE interaction.