A comparison of the inhibitory effects of prostacyclin and carbacyclin on platelet adhesion to collagen

The effect of carbacyclin, a chemically stable analogue of prostacyclin (PGI₂), on the adhesion of platelets to collagen has been examined. The compound was compared to PGI₂ which is unstable and rapidly hydrolysed to the inactive derivative, 6-oxo-PGF_1α. The adhesion of ¹¹¹Indium-labelled human plateles to collagen in the absence of platelet aggregation and secretion was measured. The cAMP level in the platelets was also monitored. Both PGI₂ and carbacyclin inhibited platelet-collagen adhesion and caused a rise in the platelet cAMP level. Carbacyclin was approximately 15-fold less effective than PGI₂, however, its effect was longer lasting, remaining constant for at least 30 minutes.     

A comparison of the inhibitory effects of melatonin and indomethacin on platelet aggregation and thromboxane release

Collagen-induced platelet aggregation and thromboxane release is inhibited, in a concentration response relationship, by preincubation of gel-filtered platelets with melatonin in the concentration range 430 nM – 4.3 mM. Inhibition of platelet aggregation and thromboxane release also occurs in the presence of indomethacin (4.3 nM – 4.3 mM), a known potent inhibitor of prostaglandin synthesis. Arachidonic acid-induced platelet aggregation and thromboxane release was inhibited in the presence of 4.0 mM melatonin. We therefore propose that inhibition of prostaglandin synthesis maybe the mechanism by which melatonin expresses its activity. Its antigonadotropic activity may result from inhibition of PGE₂ synthesis in the hypothalamus and median eminence.     

Effect of prostacyclin (PGI2) on platelet adhesion to rabbit arterial subendothelium.

The effect of prostacyclin on platelet aggregation and adhesion was investigated in everted pieces of rabbit abdominal aorta, from which the endothelium had previously been removed. Citrated human blood, to which different, concentrations of prostacyclin (0.1-100 ng/ml) were added, was perfused through the vessels, after which sections were examined and evaluated by light microscopy. Prostacyclin inhibited thrombus formation at concentrations greater than 0.1 ng/ml, whereas 20 ng/ml were required to reduce the amount of adhesion to the subendothelial surface. Thus prostacyclin prevents thrombus formation at much lower concentrations than are needed to inhibit platelet-vessel wall interaction.     

Differential Involvement of ERK2 and p38 in platelet adhesion to collagen

We investigated the role of two MAP kinases, ERK2 and p38, in platelet adhesion and spreading over collagen matrix in static and blood flow conditions. P38 was involved in collagen-induced platelet adhesion and spreading in static adhesion conditions, whereas ERK2 was not. In blood flow conditions, with shear rates of 300 or 1500 s(-1), ERK2 and p38 displayed differential involvement in platelet adhesion, depending on the presence or absence of the von Willebrand factor (vWF). Low collagen coverage densities (0.04 microg/cm2) did not support vWF binding. During perfusions over this surface, platelet adhesion was not affected by the inhibition of ERK2 phosphorylation by PD 98059. However, abolishing p38 activation by SB 203580 treatment reduced platelet adhesion by 67 +/- 9% at 300 s(-1) and 56 +/- 2% at 1500 s(-1). In these conditions, the p38 activity required for platelet adhesion depends on the alpha2beta1 collagen receptor. At higher collagen coverage densities (0.8 microg/cm2) supporting vWF binding, the inhibition of ERK2 activity by PD 98059 decreased adhesion by 47 +/- 6% at 300 s(-1) and 72 +/- 3% at 1500 s(-1), whereas p38 inhibition had only a small effect. The ERK2 activity required for platelet adhesion was dependent on the interaction of vWF with GPIb. In conclusion, ERK2 and p38 have complementary effects in the control of platelet adhesion to collagen in a shear stress-dependent manner.     

Determination of platelet adhesion to collagen and the associated formation of phosphatidic acid and calcium mobilization

A method was developed to study the adhesion of platelets to fibrillar collagen at 37 degrees C in the absence of aggregation. Human platelets were labeled with [3H]-oleic acid, gel-filtered, and incubated with collagen in the presence of receptor antagonists to thromboxane A2, 5-hydroxytryptamine, and platelet-activating factor, as well as a fibrinogen/fibronectin inhibitor and an ADP-removing system. Those platelets that adhered to collagen were separated from those that did not by filtration through a 10-microns nylon mesh and the extent of platelet adhesion was quantitated by determination of the radioactivity retained by the mesh. The extent of platelet adhesion was proportional to the amount of collagen added up to 100 micrograms/ml and was essentially complete by 1 min. At least 80-90% of the platelets were capable of adhering to collagen. Adhesion was potentiated by the presence of extracellular Mg2+ and this potentiation was inhibited by extracellular Ca2+. Phosphatidic acid increased markedly in those platelets that adhered to collagen and this was associated with increases in cytosolic free Ca2+ levels that could be detected using the fluorescent Ca2+ indicator fura-2.     

Effect of prostacyclin (PGI2) on platelet adhesion to rabbit arterial subendothelium

The effect of prostacyclin on platelet aggregation and adhesion was investigated in everted pieces of rabbit abdominal aorta, from which the endothelium had previously been removed. Citrated human blood, to which different concentrations of prostacyclin (0.1–100 ng/ml) were added, was perfused through the vessels, after which sections were examined and evaluated by light microscopy. Prostacyclin inhibited thrombus formation at concentrations greater than 0.1 ng/ml, whereas 20 ng/ml were required to reduce the amount of adhesion to the subendothelial surface. Thus prostacyclin prevents thrombus formation at much lower concentrations than are needed to inhibit platelet-vessel wall interaction.     

Prostaglandins: specific inhibition of platelet adhesion to collagen and relationship with cAMP level

The effect of 3 prostaglandins (PG's) (I2, D2 and E1) on the adhesion of platelets to purified type III collagen has been investigated. A quantitative method for a specific evaluation of the adhesion has been applied and has revealed an inhibition of adhesion by low concentrations (10(-10)M) of PGs added before collagen; the effect varied as a function of the dose of PGs (maximum at 10(-6)M) which also induced an increase in the level of platelet cAMP. The inhibition of adhesion and the elevation of platelet cAMP followed the same time course and were either of short duration (rapid decrease in the induced effects after 15 and 45 seconds in the case of PGE1) or longer lasting (maximum effect maintained for 5 minutes in the case of PGI2 and D2). These effects were potentiated by a phosphodiesterase inhibitor such as theophylline (10(-3)M). The addition of PGs after collagen resulted in a reduction of the enhancement of cAMP, associated with a decrease in the inhibition of adhesion. Moreover, the addition of exogenous cAMP (dibutyryl N6-02' cAMP) induced a comparable inhibition. A correlation between the adhesion of platelets to collagen and the level of either endogenous or exogenous cAMP has been established. The PGs also inhibited the platelet release reaction from the alpha granules (beta TG) and the dense bodies. (5-HT and ADP). A greater inhibition of release than of adhesion was observed for the same doses of PGs added.     

Distinct determinants on collagen support alpha 2 beta 1 integrin-mediated platelet adhesion and platelet activation.

Recent studies have revealed that the sequence of amino acids asp-gly-glu-ala represents an essential determinant of the site within the alpha 1(I)-CB3 fragment of collagen recognized by the alpha 2 beta 1 integrin cell surface collagen receptor (Staatz et al., 1991). Studies employing chemical modifications of collagen amino acid side chains confirm both the essential nature of the acidic side chains of aspartic acid and glutamic acid residues and the nonessentiality of lysine epsilon-amino groups in supporting adhesion mediated by the alpha 2 beta 1 integrin. The approach also indicates the presence of a distinct determinant on collagen separate from the alpha 2 beta 1 recognition site that contains essential lysine side chains and that is necessary for subsequent interactions with the platelet surface that give rise to collagen-induced platelet activation and secretion. The two-step, two-site model for cellular signaling involving both an integrin and a signal-transducing coreceptor suggested by these data may be common to other integrin-mediated processes.     

The human fibroblast class II extracellular matrix receptor mediates platelet adhesion to collagen and is identical to the platelet glycoprotein Ia-IIa complex

A monoclonal antibody, P1H5, to the human fibroblast class II extracellular matrix receptor (ECMR II) specifically inhibits human fibroblast adhesion to collagen and immunoprecipitates a cell surface receptor containing an alpha and beta subunit of approximately 140 kilodaltons each (Wayner, E. A., and Carter, W. G. (1987) J. Cell Biol. 105, 1873-1884). We report here that P1H5 also specifically inhibits adhesion of unactivated human platelets to type I and III collagens, but not to fibronectin. Immunoprecipitation of the class II ECMR from Triton X-100 detergent lysates of platelets, after cell surface iodination, identified the platelet collagen receptor. Peptide mapping confirmed that the II alpha and II beta subunits immunoprecipitated from platelets are structurally homologous with those derived from fibroblasts. The platelet ECMR II alpha and -beta subunits comigrate with platelet membrane glycoproteins Ia and IIa, respectively, on two-dimensional nonreduced-reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. These results indicate that platelet and fibroblast adhesion to collagen are both mediated by a similar receptor and that the alpha and beta subunits of this receptor are identical to platelet membrane glycoproteins Ia and IIa, respectively. Although glycoprotein Ia has been previously implicated as a collagen binding protein, our results are the first direct evidence that platelet glycoprotein Ia is associated with glycoprotein IIa in a heterodimer complex and that this complex, by mediating platelet attachment, is an actual receptor for platelet adhesion to collagen.     

Comparison in anesthetized dogs of the anti-aggregatory and hemodynamic effects of prostacyclin and a chemically stable prostacyclin analog, 6a-carba-PGI2 (carbacyclin)

The intravascular anti-aggregatory and systemic and hemodynamic effects of prostacyclin and carbacyclin were compared by intravenous infusion in pentabarbital anesthetized dogs. Ten times as much carbacyclin was needed to produce comparable inhibition of platelet aggregation in the lumen of partially obstructed circumflex coronary arteries. These doses of carbacyclin caused similar decreases in total peripheral resistance as equi-effective anti-aggregatory doses of prostacyclin. There was a trend for the decrease in blood pressure with carbacyclin to be less than that produced by equi-effective anti-aggregatory doses of prostacyclin because carbacyclin caused somewhat greater increases in cardiac output. Changes in heart rate were similar with both substances. During carbacyclin and prostacyclin infusion resistance in normal (unobstructed) coronary arteries decreased. Both substances had comparable effects on pulmonary vascular resistance, right atrial pressure and left ventricular dp/dt at equivalent anti-aggregatory doses both before and after atropine (1 mg/kg) and hexamethonium (5 mg/kg). During 5 to 6 hour infusions of carbacyclin there was no evidence of desensitization of dog platelets to the anti-aggregatory activity. These results show that carbacyclin has a similar spectrum of activity as prostacyclin and is about one-tenth as potent.     

Effect of prostacyclin analogue (carbacyclin) on the interaction of platelets with different collagen substrates. Inhibition of camp inccrease by collagens type I, III and IV

We investigated the effects of a stable prostacyclin analogue, carbacylcin, on the interaction of platelets with collagen substrates differing in thier ability to activate platelets: human collagens type I. III, IV and V (CI, CIII, CIV and CV), and commercial calf skin collagen type I (CSC). The total adhension was measured using ⁵¹Cr-labelled platelets, and quantitative morphometry of adherent platelets was performed by scanning electron microscopy (SEM). Carbacyclin in the concentrations inducing a 10-fold rise in platelets cAMP did not effect the adhension of platelets to weak substrates, CV and CSV, but reduced the adhesion to strong substrates, CIV and *by 49%) and CI/CIII (by 78%), which stimulated massive spreading and formation of surface-boud aggregates respectively. Carbacyclin inhibited all morphological manifestations of platelet activation associated with adhension: conversion of native discoid platelets to spherical ones on CSC; massive spreading on CIV; and aggregate formation on CI/CIII. Massive spreading and aggregation on a weak substrate (CSC) stimulated by arachidonic acid and thrombin was also inhibited by carbacyclin. Under the same concentration of angonists aggregation of platelets was more sensitive to the action of carbacyclin, than spreading. Strong collagen substrates CI, CIII and CIV, but not CV and gelatin, inhibited the carbacyclin-induced rise in platelet cAMP.     

The inhibitory effects of okadaic acid on platelet function.

Okadaic acid (OA), a potent inhibitor of protein phosphatases type 1 and type 2A, inhibited thrombin-induced platelet aggregation (IC50 = 0.8 microM), [14C]serotonin release and increase in intracellular Ca2+ ([Ca2+]i) in the same dose dependence. In the absence of thrombin OA increased the phosphorylation of 50-kDa protein and 20-kDa myosin light chain (MLC20). The 50-kDa protein phosphorylation was accomplished within a shorter time period and at a lower concentration than was the MLC20. OA decreased the thrombin-induced phosphorylation of 47-kDa protein and MLC20, although phosphorylation of MLC20 reincreased at higher concentrations of OA (5-10 microM). Since type 2A phosphatase is more sensitive to OA than type 1, these results suggest that type 2A phosphatases are involved in the regulation of Ca2+ signaling in thrombin-induced platelet activation.     

Effects of prostacyclin analogue (carbacyclin) on the interaction of platelets with different collagen substrates. Inhibition of cAMP increase by collagens type I, III, and IV

We investigated the effects of a stable prostacyclin analogue, carbacyclin, on the interaction of platelets with collagen substrates differing in their ability to activate platelets: human collagens type I, III, IV and V (CI, CIII, CIV and CV), and commercial calf skin collagen type I (CSC). The total adhesion was measured using 51Cr-labelled platelets, and quantitative morphometry of adherent platelets was performed by scanning electron microscopy (SEM). Carbacyclin in the concentrations inducing a 10-fold rise in platelet cAMP did not affect the adhesion of platelets to weak substrates, CV and CSC, but reduced the adhesion to strong substrates, CIV (by 49%) and CI/CIII (by 78%), which stimulated massive spreading and formation of surface-bound aggregates respectively. Carbacyclin inhibited all morphological manifestations of platelet activation associated with adhesion: conversion of native discoid platelets to spherical ones on CSC; massive spreading on CIV; and aggregate formation on CI/CIII. Massive spreading and aggregation on a weak substrate (CSC) stimulated by arachidonic acid and thrombin was also inhibited by carbacyclin. Under the same concentration of agonists aggregation of platelets was more sensitive to the action of carbacyclin, than spreading. Strong collagen substrates CI, CIII and CIV, but not CV and gelatin, inhibited the carbacyclin-induced rise in platelet cAMP.     

A comparison of prostacyclin and heparin on hemodialyses in dogs

A comparison of the efficacy of prostacyclin and herapin as anticoagulants during hemodialysis in uremic dogs was evaluated. Prostacyclin was infused continuously into the arterial limb of the dialyzer (100 ng/kg/min) while heparin was administered as a 5000 unit bolus at the beginning of dialysis. Prostacyclin and heparin were alternated and a total of 4 dialyses/dog were performed. Parameters followed to assess the efficiency of dialyses included: dialysance (ml/min) of urea, creatinine, ultrafiltration rate (ml/hr), residual volume and platelet count (% of baseline). A lesser degree of hemodialysis associated thrombocytopenia, and a higher ultrafiltration rate were observed with prostacylcin. These studies demonstrate that prostacyclin can be used alone as an anticoagulant in a uremic dog model, and in contrast to heparin it maintains the platelet count and improves the ultrafiltration rate during hemodialysis.     

Relative effects of flurbiprofen on platelet 12-hydroxy-eicosatetraenoic acid and thromboxane A2 production: influence on collagen-induced platelet aggregation and adhesion

Flurbiprofen has been shown to inhibit cyclo-oxygenase metabolism of arachidonic acid to thromboxane A2 (TxA2), resulting in the inhibition of platelet aggregation. Recently, our laboratory reported that the "irreversible" phase of platelet aggregation and adhesion were regulated, in part, by the lipoxygenase metabolism of arachidonic acid to 12-hydroxy-eicosatetraenoic acid (12-HETE) in platelets, and that selective inhibition of one enzyme i.e. either cyclo-oxygenase or lipoxygenase, resulted in paradoxical effects on the metabolism of arachidonic acid and platelet response related to the other pathway. Therefore, we performed experiments to assess the relative effects of flurbiprofen on TxA2 and 12-HETE synthesis, and on collagen-induced platelet aggregation and platelet adhesion to collagen-coated surfaces. "Irreversible" collagen-induced platelet aggregation was only partially inhibited by pre-incubation with 1 x 10(-6) M flurbiprofen, while TxA2 production was elevated and 12-HETE production was maximally inhibited in these platelets. At this concentration of flurbiprofen (1 x 10(-6)M), collagen-induced platelet adhesion was also reduced by 50%. At higher concentrations of flurbiprofen, both platelet aggregation and adhesion were further reduced, with a corresponding inhibition of TxA2 production. Thus it appears that the lipoxygenase pathway of arachidonic acid metabolism in platelets is not only inhibited by flurbiprofen, but is more sensitive to inhibition by flurbiprofen than the cyclo-oxygenase pathway. This differential effect of flurbiprofen on arachidonic acid metabolism in the platelet is related to differential effects on platelet function.     

Facile platelet adhesion to collagen requires metabolic energy and actin polymerization and evokes intracellular free calcium mobilization

The attachment of platelets to collagen-coated microtiter plates at 20 degrees C was inhibited strongly by depletion of metabolic energy or by addition of cytochalasins and was slightly inhibited by the intracellular Ca2+ chelator BAPTA. In keeping with their respective potencies as inhibitors of actin polymerization, cytochalasins D and H were the most potent inhibitors of adhesion, while cytochalasin B was the least potent. Energy depletion, cytochalasin D or, to a much lesser extent, BAPTA also inhibited platelet adhesion to collagen in a suspension assay system at 37 degrees C. Collagen-induced platelet cytosolic Ca2+ mobilization was inhibited up to 70% by cytochalasin D and abolished by energy depletion or BAPTA. Elevation of intracellular platelet calcium by treatment with ionomycin had little effect on platelet adhesion to collagen. We propose that rapid platelet spreading along collagen fibers is both energy- and actin-dependent and necessary to produce maximal adhesion needed to elicit Ca2+ mobilization required for subsequent responses.