The effects of intravenous prostacyclin in a model of microsurgical thrombosis

Numerous agents have been administered in an attempt to achieve specific biochemical antiplatelet activity. A model of microsurgical trauma was utilized to create a nonocclusive thrombus, similar to what occurs in the postoperative period. Prostacyclin (PGI2) was given in a high intravenous dose which caused in vitro inhibition of platelet aggregation in rats and rabbits. Although hematological and cardiovascular side effects of PGI2 were tolerated, in vivo platelet thrombus formation persisted and constituted 25-75% of the postoperative thrombus. Even though platelets were inhibited by PGI2, other significant stimuli remained at the site of injury for activation and participation of platelets in the formation of a thrombus.     

Inferior vena cava replacement: the role of antiplatelet therapy

To evaluate the effect of ibuprofen on early thrombus formation following inferior vena cava (IVC) replacement, a 4-cm segment of IVC was replaced with a 5-cm (10-mm-i.d.) segment of reinforced polytetrafluoroethylene (PTFE) graft in 12 dogs. Autologous platelets and canine fibrinogen were labeled with 111In and 125I, respectively, and injected into each animal 24 hr prior to vena cava replacement. Six dogs served as controls and six were treated with 12.5 mg/kg ibuprofen intravenously 1 hr preoperatively. All dogs were heparinized with 100 U/kg intravenous heparin prior to crossclamping the IVC: heparin was not reversed at the end of the procedure. Three hours after normal circulation was restored, the grafts were removed and counts of radioactivity made. All grafts were patent. The mean platelet count for the control group was 12.8 X 10(6)/mm2, while in the grafts from the treated group it was 0.960 X 10(6)/mm2. The decreased platelet deposition was significant in all graft segments (P less than 0.01). Fibrin deposition was reduced from 3.38 micrograms/mm2 to 0.25 micrograms/mm2 (P less than 0.01) by ibuprofen. Although fibrin and red blood cells are the major constituents of venous thrombi, platelet aggregation appears to play an important role if prosthetic material is implanted into the venous system. Ibuprofen not only reduced platelet deposition by 13.5-fold, but also reduced fibrin deposition by 13.5-fold. The ratio of platelets to fibrin in control and treated animals was similar (3.84 and 3.79, respectively). These data suggest that antiplatelet medication combined with heparin therapy might decrease early thrombus formation in venous prostheses.     

Scanning electron microscopy of crush/avulsion arterial trauma:effect of heparin and aspirin administration

A model of vascular trauma and subsequent reanastomosis with poor postoperative likelihood of patency was developed in the rat femoral artery. Patency rates were significantly improved with intravenous heparin, intragastric aspirin, and both agents together. Heparin yielded higher patency than aspirin. Intimal surfaces of the vessels at various postoperative intervals were observed with the scanning electron microscope. More fibrin accumulation was seen in the aspirin-treated animals, while more platelet aggregation was found in the heparin-treated group. Both platelet aggregation and fibrin strand development appeared retarded with both agents administered. All groups demonstrated good healing of the ruptured intimal surfaces, beginning at 2 days postoperatively. It is concluded that fibrin strand development is a more significant factor in microvascular occlusion than platelet aggregation.     

Effect of flunarizine on arterial thrombosis after endarterectomy in rats

Unilateral carotid endarterectomy was performed microsurgically in 60 rats. The effects of perioperative administration of aspirin and flunarizine, a Ca2+ entry blocker, on thrombus formation were examined 24 hours after endarterectomy. An untreated group of endarterectomized rats served as controls. Platelet adhesion, blood cells, and fibrin cords were observed at the cut edge of the intima as well as in the endarterectomized area in all three groups. However, these findings were significantly more extensive in the control group than in the drug-treated groups. Flunarizine was as effective as aspirin in inhibiting platelet adhesion and fibrin cord formation. Both drugs significantly increased the patency rate after carotid endarterectomy.     

White clot syndrome. Peripheral vascular complications of heparin therapy.

Heparin sodium-induced thrombosis is insidious and difficult to diagnose. If untreated, it results in death or major amputation. We have treated seven patients with thromboses resulting from platelet aggregation induced by heparin. Four patients had acute arterial ischemia of the lower extremity, venous gangrene developed in two, and one patient had an occluded autogenous vein femoral popliteal bypass in the immediate postoperative period. The platelet count was noticeably reduced in affected patients. White platelet thrombi were noted in four patients, three of whom had acute arterial occlusion. A white thrombus was the cause of immediate failure of a femoral popliteal graft. Electron microscopic examination of these thrombi demonstrated predominantly fibrin platelet aggregates with an occasional entrapped WBC and a rare RBC. All patients receiving heparin therapy must have platelet counts performed regularly. If thrombocytopenia is detected, platelet aggregation studies are indicated. When abnormal platelet aggregation is noted, heparin therapy should be reversed with protamine sulfate and the patient treated with low-molecular-weight dextran and warfarin sodium.     

Thrombus formation on polytetrafluoroethylene surfaces: the importance of von Willebrand factor

The importance of von Willebrand factor (vWf) in the formation of platelet-fibrin thrombi on expanded polytetrafluoroethylene (ePTFE) surfaces was studied in an in vitro system, perfusing non-anticoagulated human blood over ePTFE grafts for 3 min at varying shear rates (100, 500 and 1500/s shear). Platelet (¹¹¹In) and fibrin (¹²⁵I) deposition was assessed on ePTFE surfaces in the presence and relative absence of vWf, achieved by use of polyclonal anti-vWf antibody (anti-vWf Ab). A total of 29 perfusions were performed. Increasing shear rate was associated with greater platelet deposition in the presence of vWf (P < 0.001). This shear-dependent rise in platelet deposition was not observed when vWf was blocked by anti-vWf Ab (P < 0.1), confirming the role of vWf in platelet deposition at high shear rates. Fibrin deposition increased with increasing shear rate in the presence of vWf (P < 0.01). Inhibiting vWf abolished the shear-dependent increase in fibrin deposition. These data suggest that vWf plays a critical role in platelet and fibrin thrombus formation on ePTFE surfaces. These effects are particularly important under conditions of high shear rate. These mechanisms may lead to the observed pathologic thrombus formation and platelet-dependent neointimal processes occurring at areas of high shear rate within the anastomotic regions of ePTFE grafts.     

Early versus delayed heparin reversal after carotid endarterectomy in the dog. A scanning electron microscopy study

The present study investigates the hematological reaction to arterial injury during the first 10 minutes after endarterectomy in dogs to determine if heparin reversal during this early period predisposes to thrombus formation. Known platelet physiology would predict that heparinization during this early period would be useful to allow a fibrin-free platelet monolayer to form. After systemic heparinization (145 mu/kg) of the experimental animals, 42 endarterectomies were performed. Blood flow was then resumed for specific periods of time, and the vessels were prepared for scanning electron microscopy. Group 1 vessels (from the unheparinized control group) revealed mural thrombus formation after 10 minutes of blood flow. Group 2 vessels revealed the progressive formation of a fibrin-free platelet monolayer after 2, 5, or 10 minutes of blood flow resumption under systemic heparinization. Group 3 arteries, harvested at 10 minutes, underwent immediate (within 1 to 2 minutes after resumption of flow) heparin reversal with protamine sulfate, and demonstrated numerous patches of fibrin covering the platelet monolayer. Group 4 arteries, studied after 3 hours of blood flow, also underwent immediate heparin reversal. Two of these seven specimens had clumps of fibrin overlying the platelet monolayer. The Group 5 vessels had heparin reversal at 10 minutes, and demonstrated no fibrin overlying the platelet monolayer after 3 hours of blood flow. This study demonstrates the formation of a fibrin-free platelet monolayer over the endarterectomized vessel wall within 10 minutes of resumption of flow under systemic heparinization. These findings suggest that heparin may safely be reversed following a carotid endarterectomy if one awaits the initial critical 10 minutes of blood flow.     

Platelet glycoprotein VI-dependent thrombus stabilization is essential for the intraportal engraftment of pancreatic islets

Platelet activation and thrombus formation have been implicated to be detrimental for intraportal pancreatic islet transplants. The platelet-specific collagen receptor glycoprotein VI (GPVI) plays a key role in thrombosis through cellular activation and the subsequent release of secondary mediators. In aggregometry and in a microfluidic dynamic assay system modeling flow in the portal vein, pancreatic islets promoted platelet aggregation and triggered thrombus formation, respectively. While platelet GPVI deficiency did not affect the initiation of these events, it was found to destabilize platelet aggregates and thrombi in this process. Interestingly, while no major difference was detected in early thrombus formation after intraportal islet transplantation, genetic GPVI deficiency or acute anti-GPVI treatment led to an inferior graft survival and function in both syngeneic mouse islet transplantation and xenogeneic human islet transplantation models. These results demonstrate that platelet GPVI signaling is indispensable in stable thrombus formation induced by pancreatic islets. GPVI deficiency resulted in thrombus destabilization and inferior islet engraftment indicating that thrombus formation is necessary for a successful intraportal islet transplantation in which platelets are active modulators.     

The Effect of Coumadin Upon Thrombus Forming on Foreign Surfaces

Experience with the Stagnation Point Flow Experiment has revealed the earliest microscopic events surrounding thrombus inception and growth on foreign surfaces in a perfectly defined flow field. Using 40 and 80 × magnification, the effects of Coumadin upon these events was studied. These events include leukocyte adherence, fibrin deposition and platelet aggregation, deviation of flow streamlines around these aggregates, and subsequent downstream thrombus growth. Surface density of leukocytes was measured as a function of time and shear, and served as an index of the quantity of thrombus on the surface, which in turn, allowed direct comparisons of thrombus growth rate between experiments. Coumadin decreases the adhesive force between leukocytes or thrombi and the foreign surface. Emboli are more frequent with Caumadin, but are smaller. Thrombus inception is not altered. These results suggest that clinical benefit obtained from use of Coumadin derives from reduction in size of emboli.     

Drug effects on platelet deposition after endothelial injury of the rabbit aorta

Factors released from platelets deposited on injured endothelium have lately been increasingly implicated in the pathogenesis of atherosclerosis and neointimal hyperplasia. Inhibition of platelet activity has therefore been postulated to protect injured vessels from progressive degenerative disease. The objective of this study was to evaluate the effectiveness of platelet inhibiting drugs in decreasing the deposition of platelets after a standardized endothelial injury of the rabbit aorta. The aortic endothelium of 53 rabbits was denuded with a balloon catheter. Morphological changes were studied with light and electron microscopy in five rabbits. The influence of ibuprofen, acetylsalicylic acid, dipyridamole, verapamil, and prostacyclin upon the deposition of indium-111-labeled autogenous platelets 1 hr after injury was evaluated in 48 animals. The morphological studies demonstrated that platelets were deposited on the denuded aorta but no major platelet aggregation or thrombus formation occurred. The radionuclide studies showed that none of the drugs tested had any significant influence on the deposition of platelets. It is concluded that platelets immediately adhere to injured vessels but that the secondary platelet aggregation is minimal if the injury is limited to the endothelium. Conventional drugs mainly affecting platelet aggregation are ineffective in these circumstances.     

Does dextran 40 reduce early graft thrombogenicity? An experimental investigation on patency and platelet deposition on prosthetic graft materials in sheep.

The mechanism by which dextran 40 reduces early graft thrombogenicity has not been fully elucidated. Dextran improves haemaodynamics, reduces platelet aggregation, alters fibrin formation and enhances thrombus lysis. In this experimental investigation on sheep using a low flow model, the thrombogenicity of various grafts was studied when either a dextran or saline infusion was given. Bilateral carotid interposition grafts with expanded polytetrafluorethylene (ePTFE) on one side and dacron on the other (random allocation) were inserted in 12 sheep. The sheep were randomly divided into two groups, one given a dextran infusion and the other saline. A tendency for improved graft patency was seen in the dextran 40 treated animals (P less than 0.05 at 3 h). However, platelet accumulation did not differ markedly between the dextran 40 and saline treated groups. On the other hand there was a clear reduction of platelet accumulation on ePTFE grafts compared to dacron grafts (P less than 0.01). A large part of the radioactivity measured from the dacron graft was located within the graft wall. Further studies to clarify the mechanism of action of dextran are needed.     

Inhibition of platelet aggregation in baboons: therapeutic implications for xenotransplantation

Methods: Drugs tested in these experiments were aurintricarboxylic acid (ATA, von Willebrand Factor-GPIb inhibitor), fucoidin (a selectin-inhibitor), 1-benzylimidazole (1-BI, thromboxane synthase antagonist), prostacyclin (PGI₂, endothelial stabilizer), heparin (thrombin antagonist), nitroprusside sodium or nicotinamide (NPN or NA, both NO-donors), and eptifibatide (EFT, GPIIb/IIIa receptor antagonist). These were infused intravenously to nine baboons. Coagulation parameters and platelet counts were monitored and baboons were observed for adverse side-effects. The efficacy of these agents in inhibiting platelet aggregation was assayed in a platelet aggregometer.
Results: Treatment with ATA and fucoidin resulted in complete inhibition of platelet aggregation but also in major perturbation of coagulation parameters. 1-BI and PGI₂ had no effect when administered alone, but in combination resulted in moderate inhibition of aggregation without disturbance in PT or PTT. NPN and NA had no substantive effects on platelet aggregation. Heparin resulted in specific inhibition of thrombin-induced platelet aggregation and, as anticipated, was associated with moderate prolongation of PTT. Importantly, EFT caused complete inhibition of platelet aggregation without changes in coagulation. Platelet counts, fibrinogen levels, and fibrinogen degradation products remained within the normal ranges in all experiments.
Conclusions: Although excellent inhibition of platelet activation was obtained with ATA and fucoidin, clinical use may be precluded by concomitant disturbances of coagulation. Combinations of heparin and EFT may prove beneficial in preventing the thrombotic disorders associated with xenograft rejection while maintaining adequate hemostatic responses. These agents are to be evaluated in our pig-to-primate xenotransplantation models.     

Nitric oxide-releasing biopolymers inhibit thrombus formation in a sheep model of arteriovenous bridge grafts

OBJECTIVES: Nitric oxide (NO), produced by normal vascular endothelial cells, reduces platelet aggregation and thrombus formation. NO-releasing biopolymers have the potential to prolong vascular graft and stent patency without adverse systemic vasodilation. METHODS: 5-mm polyurethane vascular grafts coated with a polymer containing the NO-donor dialkylhexanediamine diazeniumdiolate were implanted for 21 days in a sheep arteriovenous bridge-graft model. RESULTS: Eighty percent (4/5) of grafts coated with the NO-releasing polymer remained patent through the 21 day implantation period, compared to fifty percent (2/4) of sham-coated grafts and no (0/3) uncoated grafts. Thrombus-free surface area (+/-SEM) of explanted grafts was significantly increased in NO-donor coated grafts (98.2% +/- 0.9%) compared with sham-coated (79.2% +/- 8.6%) and uncoated (47.2% +/- 5.4%) grafts ( P = .00046). Examination of the graft surface showed no adherent thrombus or platelets and no inflammatory cell infiltration in NO-donor coated grafts, while control grafts showed adherent complex surface thrombus consisting of red blood cells in an amorphous fibrin matrix, as well as significant red blood cell and inflammatory cell infiltration into the graft wall. CONCLUSION: In this study we determined that local NO release from the luminal surface of prosthetic vascular grafts can reduce thrombus formation and prolong patency in a model of prosthetic arteriovenous bridge grafts in adult sheep. These findings may translate into improved function and improved primary patency rates in small-diameter prosthetic vascular grafts.     

Reduction of dialyzer fibrin deposition with sulphinpyrazone

To determine whether sulphinpyrazone reduces thrombus formation within artificial kidneys, dialyzer 125I-fibrinogen and platelet and fibrinogen levels during dialysis were compared during a non-treatment control period and while patients were receiving sulphinpyrazone. Mean fibrin deposition within the dialyzers, measured as gram X 10(-3) of clottable fibrinogen, was significantly less during sulphinpyrazone treatment (2.5) than during the control period (5.3). Arterial blood platelet counts and plasma fibrinogen levels during dialysis were higher on treatment despite similar predialysis values during control and treatment periods. The results indicate that sulphinpyrazone reduces fibrin formation within artificial kidneys and, since the reduction in deposition of fibrin alone is insufficient to explain the higher plasma fibrinogen levels during treatment with sulphinpyrazone, suggests that this therapy reduces fibrinogen consumption within the patient during hemodialysis.     

Microthrombus formation on hemodialysis membranes: a placebo controlled randomized trail of two doses of Indobufen

The role of Indobufen in preventing the formation of microthrombi on hemodialysis membranes has been investigated in 18 patients in a placebo controlled randomized double-blind cross-over study. All patients had been on regular maintenance hemodialysis for at least 3 months. Indobufen was given as 100 mg b.d. and 200 mg b.d. each for a 7 day period with a 7 day wash-out period between the treatments. Both Indobufen regimens prevented the fall in platelet count, reduced the increase in plasma BTg levels during dialysis, increased the post dialysis plasma heparin levels (p less than 0.05) and inhibited pre-dialysis platelet aggregation with collagen (p less than 0.05), when compared with placebo treatment. Scanning electron microscopy demonstrated minimal fibrin and reduced platelet deposition following Indobufen treatment. There was no difference in the effect of 100 mg b.d. and 200 mg b.d. Indobufen doses. The drug was well tolerated, despite the relatively high levels measured, only one patient withdrew because of side effects. This study indicates that Indobufen when added to a routine hemodialysis treatment schedule, can significantly reduce platelet activation and the thrombus formation on the hemodialysis membranes.     

A preparation to study simultaneous arterial and venous thrombus formation in rabbits.

Injury to an artery induces formation of a platelet-rich thrombus, while stasis or trauma to a vein induces a fibrin-rich thrombus. We have implemented preparations for evolving both platelet-rich and fibrin-rich thrombi simultaneously in rabbits for use to define the efficacy of novel antithrombotic agents. For platelet-rich thrombosis, a carotid artery and contralateral jugular vein were dissected and an arteriovenous shunt inserted distally to prevent cerebral infarction during thrombus formation. The shunted artery was then instrumented with a proximal Doppler probe for measuring flow velocity and a distal transluminal needle electrode. Electrical injury to the artery was induced by application of 250 microA of anodal current to the indwelling needle electrode. Thrombotic occlusion was consistently observed within 60 min, permitting measurements of the effects on the incidence and time of occlusion of antithrombotic agents administered over 2 h. For fibrin-rich thrombosis, an external jugular vein was dissected, including the distal bifurcation. One of the branches was catheterized and a copper wire with cotton threads attached was advanced through the catheter into the superior vena cava, allowing exposure of the threads to flowing blood. A 25- to 30-mg thrombus was formed within 2 h, permitting reliable measurements of effects on thrombus weight of antithrombotic agents administered during this interval. Implementing both arterial and venous thrombosis simultaneously did not change measurements compared with either method alone. This approach may facilitate recognition of differences in efficacy of selected agents against thrombi of diverse composition.     

Prevention of Platelet Deposition and Thrombus Formationon Hemodialysis Membranes: A Double-Blind Randomized Trial of Aspirin and Dipyridamole

Abstract: A double-blind crossover study comparing low-dose aspirin (ASA) and dipyridamole (DPM) (100 mg ASA+75 mg DPM, t.d.s.), high-dose ASA and DPM (300 mg ASA+75 mg DPM, t.d.s.), and placebo on platelet deposition and thrombus formation on hemodialysis membranes was undertaken in 17 long-term dialysis patients. The high-dose combination significantly reduced the fall in platelet count during dialysis and also significantly increased postdialysis heparin concentrations. Scanning electron microscopy of the Cuprophan membranes showed a reduction in platelet desposition and fibrin formation during both treatment schedules, but this was most marked with the high-dose combination. The results of this study indicate that there is a graded response to combined ASA-DPM treatment and that this can significantly reduce platelet consumption and contact activation of fibrin during hemodialysis with Cuprophan membranes.     

The Vicious Cycle of Nonhealing Neointima in Fabric Vascular Prostheses

Abstract: Delayed neointimal healing of a fabric vascular prosthesis was investigated in an animal study focusing on the relationship between red thrombus, fibrinolysis, and endothelialization on the luminal surface. Fabric vascular prostheses were implanted into the descending aortas of 72 dogs. Fifty-nine grafts were explanted from 1 h to 1,705 days after implantation. One hour after implantation, the graft wall was red in color due to fresh thrombus; however, at 1 day the luminal surface became white. Red thrombus reappeared at 1 week and remained present in the long-term. Microscopically the initial red thrombus contained numerous erythrocytes. The white thrombus at 1 day was composed of a dense fibrin network without erythrocytes. At 2 days numerous lacunae appeared in the fibrin layer, and at 3–5 days cavernae and low density fibrin areas were present secondary to fibrinolysis. These areas allowed the blood components to infiltrate into the fibrin layer, and as a result red thrombus reformed within it. The thrombi on the luminal surface in the long-term was always red in color and composed of complicated, multiple stages of thrombus formation, i.e., fresh thrombus with erythrocytes, dense fibrin without erythrocytes, low fibrin density areas, lacunae and cavernae in the fibrin layer, and blood component infiltration into these spaces. Thrombus was always newly formed and present, and involuted in parallel due to fibrinolysis, suggesting that these phenomena perpetuated in a vicious cycle. However, at the anastomoses fibrinolysis was present, but blood component infiltration was prevented by the endothelial cell lining. These results suggest that endothelialization may arrest the vicious cycle of non-healing neointima in fabric vascular prostheses.     

Studies on microcirculation injury of skeletal muscle due to tourniquet-induced ischemia

Blood flow does not return to all areas after long period of ischemia produced by tourniquet application. The cause of this "no-reflow phenomenon" is still a matter of controversy. The purpose of this study was to investigate the relationship between thrombus formation and the no-reflow phenomenon by comparing the change of platelet function and the findings of vessel lumen observed by scanning electron microscopy. Through the results of a microangiogram, the author confirmed that the no-reflow phenomenon occurred on the first day and disappeared on the fifth day after the tourniquet was released. Mean volume, maximum aggregation and malondialdehyde of platelets decreased at two hours to one day after tourniquet release due to the consumption of larger platelets. There were irregular endothelia and a mural thrombus in the veins of the ischemic leg on the first day following release of the tourniquet. These findings suggest that thrombus formation plays an important role in the development of the no-reflow phenomenon.     

Low dose heparin therapy: in vitro verification of antithrombotic effect

Low dose heparin therapy has been used routinely for prophylaxis of deep venous thrombosis, yet in vitro data regarding its antithrombotic effects are sparse. The effects of heparin on venous thrombus formation were studied in an in vitro perfusion system. Fresh blood collected from human volunteers was treated with varying heparin doses and perfused at a shear rate of 100 sec-1 over everted, injured porcine vein segments, simulating conditions in the venous circulation. Platelet and fibrin deposition were measured by use of indium 111 and iodine 125 radiolabels, respectively. The effects of heparin on the intrinsic coagulation cascade were monitored by the activated clotting time. Increasing doses of heparin resulted in significant reductions in fibrin and platelet deposition (ANOVA F = 2.67 and 3.17, respectively, p less than 0.05). At a dose of only 0.19 USP units/ml blood, equivalent to a 1000 unit bolus of heparin in a 70 kg man, a noticeable reduction in both fibrin and platelet deposition was observed without an increase in the activated clotting time. These data confirm the antithrombotic effects of heparin at low dose ranges and may explain the clinically observed phenomenon of deep venous prophylaxis without an appreciable alteration in the conventional coagulation assays.