Impact of a surgical site infection reduction strategy after colorectal resection.

AIM: This study was performed to determine the impact of a surgical site infection (SSI) reduction strategy on SSI rates following colorectal resection. METHOD: American College of Surgeons National Surgical Quality Improvement Program (NSQIP) data from 2006-14 were utilized and supplemented by institutional review board-approved chart review. The primary end-point was superficial and deep incisional SSI. The inclusion criterion was colorectal resection. The SSI reduction strategy consisted of preoperative (blood glucose, bowel preparation, shower, hair removal), intra-operative (prophylactic antibiotics, antimicrobial incisional drape, wound protector, wound closure technique) and postoperative (wound dressing technique) components. The SSI reduction strategy was prospectively implemented and compared with historical controls (pre-SSI strategy arm). Statistical analysis included Pearson's chi-square test, and Student's t-test performed with spss software. RESULTS: Of 1018 patients, 379 were in the pre-SSI strategy arm, 311 in the SSI strategy arm and 328 were included to test durability. The study arms were comparable for all measured parameters. Preoperative wound class, operation time, resection type and stoma creation did not differ significantly. The SSI strategy arm demonstrated a significant decrease in overall SSI rates (32.19% vs 18.97%) and superficial SSI rates (23.48% vs 8.04%). Deep SSI and organ space rates did not differ. A review of patients testing durability demonstrated continued improvement in overall SSI rates (8.23%). CONCLUSION: The implementation of an SSI reduction strategy resulted in a 41% decrease in SSI rates following colorectal resection over its initial 3 years, and its durability as demonstrated by continuing improvement was seen over an additional 2 years.

The metabolism of phosphoinositide-derived messenger molecules.

The phosphoinositides are minor phospholipids present in all eukaryotic cells. They are storage forms for messenger molecules that transmit signals across the cell membrane and evoke responses to extracellular agonists. The phosphoinositides break down to liberate messenger molecules or precursors of messenger molecules. Many different compounds are formed, although the functions of only a few are understood. Recent studies elaborating the pathways for formation of products from phosphoinositides and the factors controlling their metabolism are summarized here.

Increased platelet deposition on atherosclerotic coronary arteries.

A ruptured atherosclerotic plaque leads to exposure of deeper layers of the plaque to flowing blood and subsequently to thrombus formation. In contrast to the wealth of data on the occurrence of thrombi, little is known about the reasons why an atherosclerotic plaque is thrombogenic. One of the reasons is the relative inaccessibility of the atherosclerotic plaque. We have circumvented this problem by using 6-microns cryostat cross sections of human coronary arteries. These sections were mounted on coverslips that were exposed to flowing blood in a rectangular perfusion chamber. In normal-appearing arteries, platelet deposition was seen on the luminal side of the intima and on the adventitia. In atherosclerotic arteries, strongly increased platelet deposition was seen on the connective tissue of specific parts of the atherosclerotic plaque. The central lipid core of an advanced plaque was not reactive towards platelets. The results indicate that the atherosclerotic plaque by itself is more thrombogenic than the normal vessel wall. To study the cause of the increased thrombus formation on the atherosclerotic plaque, perfusion studies were combined with immunohistochemical studies. Immunohistochemical studies of adhesive proteins showed enrichment of collagen types I, III, V, and VI, vitronectin, fibronectin, fibrinogen/fibrin, and thrombospondin in the atherosclerotic plaque. Laminin and collagen type IV were not enriched. von Willebrand Factor (vWF) was not present in the plaque. The pattern of increased platelet deposition in serial cross sections corresponded best with areas in which collagen types I and III were enriched, but there were also areas in the plaque where both collagens were enriched but no increased reactivity was seen. Inhibition of platelet adhesion with a large range of antibodies or specific inhibitors showed that vWF from plasma and collagen types I and/or III in the plaque were involved. Fibronectin from plasma and fibronectin, fibrinogen, laminin, and thrombospondin in the vessel wall had no effect on platelet adhesion. We conclude that the increased thrombogenicity of atherosclerotic lesions is due to changes in quantity and nature of collagen types I and/or III.

Development of a potent thrombin receptor ligand.

The N-terminal thrombin receptor peptide H-Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe-OH (1) fully activates the thrombin receptor with an EC50 of 10 microM. Structural features in the tetradecapeptide which are responsible for receptor activation have been elucidated. Agonist potency has been enhanced 1000-fold with the design of the shortened peptide H-Ala-Phe(p-F)-Arg-Cha-HArg-Tyr-NH2 (56). This analog exhibits an EC50 of 0.01 microM and is the most potent agonist for receptor activation reported to date. The monoiodinated derivative H-Ala-Phe(p-F)-Arg-Cha-HArg-Tyr(3-I)-NH2 (59) exhibits an EC50 of 0.03 microM, a level sufficient for development of a radioligand.

Prevention of reocclusion by MCI-9038, a thrombin inhibitor, following t-PA-induced thrombolysis in a canine model of femoral arterial thrombosis.

We compared a selective thrombin inhibitor (MCI-9038; Argatroban), a thromboxane A2 (TXA2) receptor antagonist (L-670,596) and a serotonin-2 receptor antagonist (ketanserin) for their ability to hasten clot lysis and delay reocclusion in a canine model of femoral arterial thrombosis. Occlusive thrombosis was induced by insertion of a thrombogenic copper coil. Femoral arterial blood flow velocity (FABFV) was monitored directly and continuously by Doppler flowmetry. Thrombolysis was induced with tissue plasminogen activator (t-PA; 0.8 mg/kg, i.v.), starting 60 min after thrombotic occlusion and continued for 90 min. Ten minutes after occlusion, dogs received an intravenous infusion of either vehicle, MCI-9038 (10 micrograms kg-1 min-1), ketanserin (0.1 mg/kg bolus plus 5 micrograms kg-1 min-1), L-670,596 (1 mg/kg bolus plus 17 micrograms kg-1 min-1) or a combination of L-670,596 and ketanserin. All infusions were discontinued 1 h after stopping the t-PA, and were followed by a 30 min observation period. The times to thrombolysis were similar for all treatments (mean +/- SEM = 47 +/- 3; all groups). MCI-9038 prevented reocclusion, defined as permanent cessation of FABFV during the hour after stopping the t-PA. All dogs receiving MCI-9038 reoccluded within 30 min after stopping its infusion (71 +/- 3 min). Reocclusion occurred in all other dogs, except one vehicle-treated dog and a second dog that received L-670,596 plus ketanserin. Vehicle-treated dogs reoccluded within 23 +/- 8 min. Reocclusion was not delayed significantly by ketanserin, L-670,596 or the combination of the two.(ABSTRACT TRUNCATED AT 250 WORDS)

Species variability in platelet and other cellular responsiveness to thrombin receptor-derived peptides.

The aggregation of platelets from a variety of animal species in response to thrombin receptor-derived activating peptides was evaluated. A series of 14-(SFLLRNPNDKYEPF), 7-(SFLLRNP-NH2), 6-(SFLLRN-HN2) or 5-(SFLLR-NH2) residue peptides, the structures of which were based on the deduced amino acid sequence of the human thrombin receptor, promoted full aggregation of platelets in plasma from humans, African Green and Rhesus monkeys, baboons and guinea pigs at 4-50 microM depending on the peptide used. Platelets in plasma from rabbit, dog, pig, and hamster underwent a shape change but failed to aggregate in response to these peptides over 3 log units of peptide up to 800 microM, despite being fully responsive to human thrombin. However, because the receptor peptides induced shape change in the platelets from these non-aggregating species, they apparently can activate some of the intracellular signaling system(s) usually initiated by thrombin in these platelets. In contrast, platelets from rats did not undergo shape change or aggregate in response to the peptides. A 7-residue receptor-derived peptide based on the deduced amino acid sequence of the clone of the hamster thrombin receptor (SFFLRNP-N2) was nearly as efficacious as the corresponding human receptor-derived 7-residue peptide to promote aggregation of human platelets. However, the hamster peptide could not promote aggregation of hamster platelets in plasma at up to 800 microM peptide, while a shape change response was elicited. Platelets from rats, rabbits and pigs also did not aggregate in response to this peptide derived from the hamster thrombin receptor, but all species except the rat underwent a shape change.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet aggregation monitored in a 96 well microplate reader is useful for evaluation of platelet agonists and antagonists.

Optimal conditions for a method to simultaneously measure aggregation in 96 samples using a microplate reader were developed. The temperature of the assay was set at 25 degrees C, the optimal platelet concentration range was determined to be from 1-3 x 10(8) per mL, the assay volume was determined to be best at 100 microL and an agitation rate of setting #5 on the vortex was found to yield the most reliable aggregation response. After these initial assay parameters were established, EC50 values for standard platelet agonists including ADP, thrombin, collagen and thrombin receptor activating peptides were determined using the plate assay and compared to those obtained by measuring light transmittance in an aggregometer. The results were quantitatively similar, and qualitatively the shapes of the aggregations as monitored by both methods were characteristic of those expected for each agonist. The use of this assay was then extended to quantitate the inhibition of aggregation by antagonists of the fibrinogen receptor as well as by an inactive thrombin receptor peptide and by antibodies against the thrombin receptor. This method provided useful data for characterization of both platelet agonists and antagonists and should be useful for future platelet aggregation studies.

Effect of inhibitors of factor Xa or platelet adhesion, heparin, and aspirin on platelet deposition in an atherosclerotic rabbit model of angioplasty injury.

Acute thrombotic reocclusion and restenosis after successful coronary angioplasty are limitations of the procedure. Although the restenotic process is not completely understood, acute platelet deposition and thrombosis are considered important initiating mechanisms. The effort to identify pharmacologic agents capable of modifying acute platelet action following mechanical injury requires an animal model mimicking the clinical pathophysiology as closely as possible. We developed a model of angioplasty-induced injury in atherosclerotic rabbit femoral arteries. Acute 111indium-labelled platelet deposition and thrombosis were assessed four hours after balloon-injury in arteries subjected to prior endothelial damage (air desiccation) and cholesterol supplementation (one month). The effects of recombinant tick anticoagulant peptide (rTAP), a blood coagulation factor Xa (fXa) inhibitor and of recombinant leech antiplatelet protein (rLAPP), a platelet adhesion inhibitor, were compared to heparin (HEP) and aspirin (ASA). Recombinant TAP and HEP, but not rLAPP or ASA, successfully prevented thrombus formation and reduced platelet deposition in balloon-injured vessel segments to levels not significantly different from those observed in the contralateral atherosclerotic non-balloon-injured vessels. Therefore, this model, incorporating balloon catheter dilation of arteries exhibiting neointimal growth and atherosclerotic plaque formation, may be useful for evaluation of possible adjunctive therapies during angioplasty.

Non-peptidic small-molecule antagonists of the human platelet thrombin receptor PAR-1.

The thrombin receptor on human platelets is the first member identified of a new family of G-protein coupled receptors referred to as protease activated receptors (PARs). These receptors are activated by a unique mechanism involving proteolytic cleavage of a portion of the extracellular domain to generate a new N-terminus which then acts as a tethered or intramolecular ligand (agonist) for the receptor. The hexapeptide SFLLRN-NH2 comprising the new N-terminus is referred to as the Thrombin Receptor Activating Peptide, or "TRAP" Thrombin is the most potent agonist for platelet aggregation and selective blockade of the intramolecular activation step without effecting the proteolytic activity of thrombin should result in moderation of platelet activation and aggregation without interfering with the other coagulation cascade effects of thrombin. Screening of combinatorial libraries identified a novel, non-peptide PAR-1 thrombin receptor antagonist. Examination of structure-activity relationships revealed that portions of the molecule could be replaced resulting in simpler molecules of lower molecular weight that were at the same time more potent. Molecules in this series were effective antagonists of TRAP-stimulated platelet activation, but had limited activity when thrombin was the agonist. Additional directed screening and subsequent lead refinement resulted in a second series of isoxazole based compounds. Some of the resultant molecules were potent PAR-1 antagonists that were effective against both TRAP- and thrombin-stimulated receptor activation. These compounds do not inhibit the proteolytic effects of thrombin but rather interfere with the intramolecular binding of the tethered ligand (SFLLRN) to the transmembrane portion of the thrombin receptor. They represent promising leads for future explorations of antithrombotic activity of thrombin receptor antagonists.

Effect of the small molecule plasminogen activator inhibitor-1 (PAI-1) inhibitor, PAI-749, in clinical models of fibrinolysis.

BACKGROUND: The principal inhibitor of fibrinolysis in vivo is plasminogen activator inhibitor-1 (PAI-1). PAI-749 is a small molecule inhibitor of PAI-1 with proven antithrombotic efficacy in several preclinical models. OBJECTIVE: To assess the effect of PAI-749, by using an established ex vivo clinical model of thrombosis and a range of complementary in vitro human plasma-based and whole blood-based models of fibrinolysis. METHODS: In a double-blind, randomized, crossover study, ex vivo thrombus formation was assessed using the Badimon chamber in 12 healthy volunteers during extracorporeal administration of tissue-type plasminogen activator (t-PA) in the presence of PAI-749 or control. t-PA-mediated lysis of plasma clots and of whole blood model thrombi were assessed in vitro. The role of vitronectin was examined by assessing lysis of fibrin clots generated from purified plasma proteins. RESULTS: There was a dose-dependent reduction in ex vivo thrombus formation by t-PA (P < 0.0001). PAI-749 had no effect on in vitro or ex vivo thrombus formation or fibrinolysis in the presence or absence of t-PA. Inhibition of PAI-1 with a blocking antibody enhanced fibrinolysis in vitro (P < 0.05). CONCLUSIONS: Despite its efficacy in a purified human system and in preclinical models of thrombosis, the current study suggests that PAI-749 does not affect thrombus formation or fibrinolysis in a range of established human plasma and whole blood-based systems.

P-selectin antagonism reduces thrombus formation in humans.

BACKGROUND: Interaction of P-selectin with its glycoprotein ligand (P-selectin glycoprotein ligand type 1) mediates inflammatory processes that may also include vascular thrombosis. Platelet P-selectin expression is increased in patients with coronary heart disease, and its antagonism represents a potential future therapeutic target for the prevention and treatment of atherothrombosis. AIM: To investigate the effects of the novel small molecule P-selectin antagonist PSI-697 on thrombus formation in humans. METHODS AND RESULTS: In a double-blind randomized crossover study, thrombus formation was measured in 12 healthy volunteers, using the Badimon ex vivo perfusion chamber under conditions of low and high shear stress. Saline placebo, low-dose (2 m) and high-dose (20 m) PSI-697 and the glycoprotein IIb-IIIa receptor antagonist tirofiban (50 ng mL(-1)) were administered into the extracorporeal circuit prior to the perfusion chamber. As compared with saline placebo, blockade of platelet glycoprotein IIb-IIIa receptor with tirofiban produced 28% and 56% reductions in thrombus formation in the low-shear and high-shear chambers, respectively. PSI-697 caused a dose-dependent, but more modest, reduction in thrombus formation. Low-dose PSI-796 (2 m) reduced total thrombus area by 14% (P = 0.04) and 30% (P = 0.0002) in the low-shear and high-shear chambers, respectively. At the high dose (20 m), PSI-697 reduced total thrombus area by 18% (P = 0.0094) and 41% (P = 0.0008) in the low-shear and high-shear chambers, respectively. CONCLUSIONS: P-selectin antagonism with PSI-697 reduces ex vivo thrombus formation in humans. These findings provide further evidence that P-selectin antagonism may be a potential target for the prevention and treatment of cardiovascular disease.

The interaction of disagregin with the platelet fibrinogen receptor, glycoprotein IIb-IIIa.

Disagregin, a 6 kDa protein isolated from salivary glands of the tick Ornithodoros Moubata, is a potent and selective inhibitor of fibrinogen dependent platelet aggregation and of the adhesion of platelets to fibrinogen (Karczewski et al. (1994) J. Biol. Chem. 269, 6702-6708). In the current study the interaction of disagregin with purified glycoprotein IIb-IIIa (GPIIb-IIIa) was examined. Biotin-labeled disagregin (b-disagregin) bound to GPIIb-IIIa immobilized on the surface of the ELISA plate. This binding was specific, dependent on divalent cations, and was blocked by the peptides fibrinogen gamma-chain fg gamma (400-411), GPIIb(296-306) and by the RGD-containing peptide, GRGDSP. Disagregin also bound to soluble GPIIb-IIIa as demonstrated in studies using the chemical crosslinker, BS3. This binding was inhibited by the peptides fg gamma (400-411) and GPIIb(296-306). In contrast to the results in the solid phase, peptide GRGDSP had no effect on the binding of b-disagregin to soluble GPIIb-IIIa. These data demonstrate that disagregin binds to GPIIb-IIIa through a mechanism distinct from that used by RGD-containing disintegrins. Further analysis of the region(s) of disagregin which bind to GPIIb-IIIa should provide useful information for molecular modeling of the fibrinogen binding site on GPIIb-IIIa and for the design of a new class of potent fibrinogen receptor antagonists.

Simple radioimmunoassay of cortisol in diluted samples of human plasma.

We describe a simple radioimmunoassay of plasma cortisol, which can be performed in 3 h and which requires no purification, heating, or refrigeration steps. The plasma proteins are inhibited through direct competition between them and the antiserum at room temperature, at which the antiserum's affinity exceeds that of the binding proteins. Plasma, diluted with water, is incubated for 3 h at room temperature with [3H]cortisol and the antiserum. We compared results with those by the usual extraction method. The correlation between methods on evaluating normal samples with one antiserum. We compared results with those by the usual extraction method. The correlation between methods on evaluating normal samples with one antiserum was r = 0.954 (P less than 0.001), and the slope was 0.661. With three other antisera it was r = 0.922 (P less than 0.001) and slope 0.644. Plasmas with abnormal protein concentrations (i.e., from preganant women, and after corticotropin administration), tested to examine the validity of the method for routine use, and to define the role of the protein carriers, showed r = 0.859 (P less than 0.001) and slope 0.726 for the four antisera used. Additional samples, assayed with diluted standards plus stripped plasma, showed a correlation with the usual extraction method of r = 0.945 (P less than 0.001) and slope 1.026.

Isolation of an inhibitor selective for collagen-stimulated platelet aggregation from the soft tick Ornithodoros moubata.

Soluble extracts from the soft tick Ornithodoros moubata were found to inhibit collagen-, ADP-, and thrombin-stimulated platelet aggregation. One inhibitory component was purified to homogeneity by a combination of gel filtration, ion-exchange, and reverse phase high pressure liquid chromatography. The purified activity, named moubatin, is a protein of molecular weight 17,000 and it inhibits the aggregation of washed human platelets stimulated by collagen with an IC50 of approximately 50 nM in the standard assay. At a concentration of moubatin that maximally inhibited collagen-stimulated platelet aggregation, no inhibition of aggregation initiated by other effectors, including arachidonic acid, thrombin, ristocetin, and the calcium ionophore A23187, was observed. Moubatin also inhibits collagen-dependent aggregation in plasma. At a higher concentration of moubatin (> 1 microM) it was also possible to demonstrate an inhibitory effect on the final extent of aggregation induced by a low concentration of ADP. Although moubatin selectively inhibits platelet activation by collagen, it has only a minimal effect on the adhesion of platelets to collagen. The amino acid sequences of peptides derived from proteolytic cleavage of moubatin suggest that moubatin is a unique protein, consistent with its novel functional activity.

Removal of extraplatelet Na+ eliminates indomethacin-sensitive secretion from human platelets stimulated by epinephrine, ADP, and thrombin.

We have previously observed that removal of extraplatelet Na+ markedly diminishes human platelet aggregation and secretion in response to epinephrine. The present studies demonstrate that this effect of the removal of extraplatelet Na+ on platelet function is not unique to activation of platelets by alpha 2-adrenergic agents but represents a phenomenon also evident for other platelet stimuli. Thus, platelet aggregation and secretion in response to maximal concentrations of ADP and lower concentrations of thrombin (less than 0.04 unit/ml) were also markedly reduced in platelets in "Na+-free" medium, suggesting that these agents share an effector mechanism that is similarly inhibited by the removal of extraplatelet Na+. In contrast, platelet aggregation and secretion in response to higher concentrations of thrombin (greater than or equal to 0.04 unit/ml) and to 0.04-1.0 microM (15S)-hydroxy-11 alpha, 9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619), an endoperoxide analog, were identical in control platelets and in those suspended in "Na+-free" medium, indicating that platelets suspended in "Na+-free" medium are functionally intact, at least in response to some stimuli. Furthermore, the observation that U46619 can elicit platelet aggregation and secretion independently of extraplatelet Na+ indicates that the loss of platelet responsiveness to epinephrine, ADP, and low concentrations of thrombin cannot be attributed to a loss of sensitivity to the stimulus-provoked secondary mediator(s) of platelet function, endoperoxides or thromboxane A2. Treatment with indomethacin to block the secondary aggregation and secretion pathways of platelets reduced the aggregatory and secretory responses of control platelets induced by epinephrine, ADP, and low concentrations of thrombin to those characteristic of platelets suspended in "Na+-free" medium. In contrast, indomethacin did not alter the functional responses induced by these agents in platelets suspended in "Na+-free" medium, suggesting that "primary" aggregation is intact but that the "secondary" aggregation and secretion mediated by arachidonic acid metabolites are eliminated by removal of extraplatelet Na+. Consistent with this interpretation is the observation that the indomethacin-insensitive aggregation and secretion induced by U46619 and higher concentrations of thrombin were retained in platelets suspended in "Na+-free" medium. Thus, the responses eliminated by removal of extraplatelet Na+ are those eliminated by treating control platelets with indomethacin, suggesting a strong link between the presence of extraplatelet Na+ and the operation of platelet function mediated by the cyclooxygenase pathway.

Protein kinase C phosphorylates human platelet inositol trisphosphate 5'-phosphomonoesterase, increasing the phosphatase activity.

Phosphoinositide breakdown in response to thrombin stimulation of human platelets results in the formation of the calcium-mobilizing messenger molecules inositol 1,4,5-trisphosphate and inositol 1,2-cyclic-4,5-trisphosphate and of diglyceride, which activates protein kinase C. We find that protein kinase C phosphorylates and thereby increases the activity of inositol 1,4,5-trisphosphate 5'-phosphomonoesterase, a phosphatase that hydrolyzes these molecules to inert compounds. The 5'-phosphomonoesterase phosphorylated using [gamma-32P]ATP comigrates on SDS-polyacrylamide gels with a protein (40 kd) phosphorylated rapidly in response to thrombin stimulation of 32PO4-labeled platelets. Peptide maps of proteolytic digests of these two phosphorylated proteins indicate that they are the same. We propose that platelet Ca2+ mobilization is regulated by protein kinase C phosphorylation of the inositol 1,4,5-trisphosphate 5'-phosphomonoesterase. These results explain the observation that phorbol ester treatment of intact human platelets results in decreased levels of inositol trisphosphate and decreased Ca2+ mobilization upon subsequent thrombin addition.

Isolation of a phosphomonoesterase from human platelets that specifically hydrolyzes the 5-phosphate of inositol 1,4,5-trisphosphate.

Platelets, and a variety of other cells, rapidly hydrolyze the phosphoinositides in response to stimulation by agonists. One of the products of hydrolysis of phosphatidylinositol 4,5-diphosphate is inositol 1,4,5-trisphosphate, which recently has been suggested to mediate intracellular Ca2+ mobilization. We have found that human platelets contain an enzyme that degrades inositol 1,4,5-trisphosphate. We have isolated this soluble enzyme and find that it hydrolyzes the 5-phosphate of inositol 1,4,5-trisphosphate (Km = 30 microM, Vmax = 5.3 microM/min/mg of protein). The products of the reaction are inositol 1,4-diphosphate and phosphate. The apparent molecular weight of the enzyme is 38,000 as determined both by gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of 2-mercaptoethanol. This enzyme is specific for inositol 1,4,5-trisphosphate. Other water soluble inositol phosphates as well as phosphorylated sugars are not hydrolyzed, while the only inositol containing phospholipid hydrolyzed is phosphatidylinositol 4,5-diphosphate at a rate less than 1% that for inositol 4,5-trisphosphate. The inositol 1,4,5-trisphosphate 5-phosphomonoesterase requires Mg2+ for activity and is inhibited by Ca2+, Ki = 70 microM. Li+, up to 40 mM, has no effect on enzyme activity. The duration and magnitude of any inositol 1,4,5-trisphosphate response in stimulated platelets may be determined by the activity of this enzyme.

Disagregin is a fibrinogen receptor antagonist lacking the Arg-Gly-Asp sequence from the tick, Ornithodoros moubata.

A platelet aggregation inhibitor was identified in the salivary gland of the tick Ornithodoros moubata and isolated by gel filtration and reverse phase high pressure liquid chromatography. The purified inhibitor is a approximately 6-kDa protein, which we have named disagregin. It inhibits ADP-stimulated platelet aggregation in plasma with an IC50 = 104 +/- 17 nM. Disagregin also inhibits platelet aggregation induced by other agonists, including collagen, epinephrine, platelet-activating factor, thrombin, and the thrombin receptor peptide SFLLRNPNDKYEPF. It does not, however, affect platelet shape change induced by these agonists or thrombin-induced dense granule release. Disagregin inhibits platelet aggregation by binding to the platelet fibrinogen receptor. 125I-Disagregin forms a specific complex with both subunits of the fibrinogen receptor, glycoproteins IIb and IIIa, in the presence of a chemical cross-linker. It binds to unstimulated platelets with a Kd = 42.5 +/- 7.5 nM (23,800 +/- 1600 sites/platelet) and to ADP-stimulated platelets with Kd = 39.4 +/- 6.6 nM (24,050 +/- 1500 sites/platelet). Unlabeled disagregin and the snake venom disintegrin echistatin both compete for this binding. Disagregin also completely blocks platelet adhesion to fibrinogen while partially inhibiting platelet adhesion to fibronectin and having little effect on platelet adhesion to collagen. Disagregin had no effect on the adhesion of human umbilical cord vein endothelial cells to fibrinogen or vitronectin. These cells lack the glycoprotein IIb-IIIa complex; therefore, this result is consistent with the ability of disagregin to bind selectively to platelet glycoproteins IIb and IIIa. Sequence analysis of disagregin revealed 60 residues composing a unique protein. Unlike other fibrinogen antagonists it does not contain the Arg-Gly-Asp cell recognition sequence or a conservative substitution, and it has no structural homology with the Arg-Gly-Asp-containing snake venom disintegrins. Thus, disagregin is unique both in structure and function and may serve as a useful tool for the design of therapeutically useful antithrombotic agents.