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Ann Thorac Surg 1998;65:352-358
© 1998 The Society of Thoracic Surgeons

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Original Articles: Cardiovascular

The Platelet Thrombin Receptor and Postoperative Bleeding

Division of Cardiothoracic Surgery, Albany Medical College, Albany, New York, USA

Dr Ferraris, 2828 1st Ave, Suite 510, Huntington, WV 25702.

Presented at the Poster Session of the Thirty-third Annual Meeting of The Society of Thoracic Surgeons, San Diego, CA, Feb 3–5, 1997.

	Abstract

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Background. We hypothesized that small amounts of thrombin desensitize the platelet thrombin receptor during cardiopulmonary bypass (CPB), resulting in postoperative platelet dysfunction and bleeding.

Methods. Seventy-nine patients were entered into a study designed to measure changes in platelet thrombin receptor function during CPB and to correlate them to postoperative bleeding. In addition to measurements of clinical blood loss, platelet function tests of aggregation, activation, and cell–cell adhesion were used. The thrombin receptor agonist peptide (TRAP) was used to activate the platelets. Flow cytometry was used to measure various platelet surface markers and platelet–white cell interactions during CPB.

Results. Compared with preoperative values, both aggregometry and flow cytometry measured a significant reduction of TRAP-induced activation immediately and up to 24 hours after CPB. The response of other activating agents returned to normal by 24 hours. Postoperatively, 8 of 79 patients required excessive blood transfusion (10 units of blood products) and had significantly decreased TRAP-induced aggregation response.

Conclusions. Our results show that (1) platelet activation, aggregation, and adhesion to leukocytes induced by TRAP are reduced after CPB, (2) decreased thrombin receptor responsiveness is associated with excessive postoperative blood loss, and (3) because the aggregation and activation responses are different for TRAP and thrombin, there may be a second thrombin receptor on platelets that is protected from damage during CPB. These results imply that prevention of the CPB-induced effects on the thrombin receptor will lessen postoperative morbidity associated with blood transfusion.

	Introduction

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Thrombin is one of several proteolytic enzymes that are generated during cardiopulmonary bypass (CPB). Active thrombin is present during CPB in spite of apparently adequate heparin levels [1] [2]. The stimulus for thrombin generation is activation of the extrinsic coagulation pathway both from contact of blood with synthetic surfaces and from exposure of blood within the surgical wound [3]. Cellular and humoral mechanisms involving thrombin play an important part in the complex molecular processes initiated by CPB.

One of the molecular mechanisms of thrombin-induced platelet activation recently has been elucidated, and one platelet thrombin receptor has been cloned [4]. Thrombin activates cells by a proteolytic mechanism that cleaves a portion of the amino terminus of a seven-transmembrane domain thrombin receptor (STTR). This proteolytic step exposes a "tethered ligand" that participates in an extracellular signaling mechanism. The tethered ligand binds to another extracellular site on the STTR and induces a cellular response by coupling with cytoplasmic G-proteins. Short peptide chains, called thrombin receptor agonist peptides (TRAPs), that duplicate the amino terminal of the tethered ligand are capable of inducing many, but not all, of the effects of thrombin [5]. They can activate platelets and mimic the effect of thrombin on the STTR [6] [7]. The stimulation of platelets using TRAPs ensures that the STTR is activated and is the likely intermediary of the platelet-specific effect.

Abnormalities of platelet function generally are considered to be a major mechanism of perioperative bleeding in patients undergoing CPB [8] [9]. It has been difficult to identify a specific, intrinsic abnormality in these platelets. Because thrombin is the preeminent platelet agonist, newer information has focused on the effect of thrombin on platelets during CPB. Recent interest in the STTR [10] caused us to evaluate the platelet responsiveness to TRAPs during CPB. We found that the thrombin receptor is dysfunctional after CPB [11] [12] [13]. This led us to the hypothesis that thrombin interaction with target systems (particularly platelets) is dysfunctional after CPB and may cause excessive postoperative bleeding. This study attempted to test this hypothesis by determining the clinical relevance of thrombin receptor–related abnormalities after CPB.

	Material and Methods

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Patient Selection
Seventy-nine patients were entered into a research protocol that was approved by the institutional review board at our institution. The study was designed to measure changes in platelet function during operations using CPB. Patients were entered into the study if they were undergoing elective or urgent cardiac procedures. Patients who were undergoing emergency procedures were excluded. Any patient older than 21 years of age was eligible for entry. Patients were required to have normal laboratory values for commonly measured hemostatic parameters, including platelet count, activated partial thromboplastin time, and prothrombin time. No attempt was made to exclude patients who were taking antiplatelet drugs before operation and, in fact, nearly every patient in the study had taken aspirin within 7 days of operation.

Materials
Adenosine diphosphate, collagen, and ristocetin were obtained from Chronolog (Havertown, PA). Both -thrombin and -thrombin were prepared by John W. Fenton, II, M.D. (Albany, NY) and TRAPs (amino acid sequence SFLLRN) were prepared by Thomas T. Andersen, M.D. (Albany, NY). GPRP (H-gly-pro-arg-pro-OH) and fluorescein isothiocyanate (FITC-celite) were obtained from Calbiochem (LaJolla, CA) and formyl-methionyleucyl-phenylalanine was obtained from Sigma (St. Louis, MO). PAC-1 murine monoclonal antibody (specific for activated platelet glycoprotein IIb/IIIa complex) was obtained from the Cell Center at the University of Pennsylvania (Philadelphia, PA). S-12 monoclonal antibody (specific for P-selectin) was a generous gift from Rodger McEver, M.D. at the University of Oklahoma (Oklahoma City, OK). CD-42b-PE (specific for platelet glycoprotein Ib/IX), nonspecific IgG-FITC, nonspecific IgG-PE, and nonspecific IgM-FITC were obtained from Immunotech (Westbrook, ME). CD-45-PerCP monoclonal antibody (specific for human leukocytes of any type) and CD-14-FITC monoclonal antibody (specific for human monocytes) were obtained from Becton-Dickinson (San Jose, CA).

Preparation of Samples
Blood samples were drawn through a flushed, indwelling arterial line during the perioperative period at the following three times: (1) before skin incision but after the induction of anesthesia, (2) 30 minutes after the reversal of heparin as judged by the presence of a normal activated clotting time, and (3) 18 to 24 hours after operation. All samples were drawn into a syringe containing sodium citrate (0.38% final concentration). The sample was placed in a plastic tube and mixed by gentle inversion. A complete blood count was obtained using a Coulter STKS (Coulter, Hialeah, FL).

Whole Blood Aggregometry
Portions of the blood were placed in cuvettes containing isotonic saline solution and warmed to 37°C before undergoing aggregometry. Standard whole blood aggregometry methods were used [14] [15]. All samples were diluted to a final platelet count of 125,000/µL by adding an appropriate volume of saline solution to the whole blood samples. Previous studies have shown that dilution of samples to a final count as low as 80,000/µL provides reproducible aggregation results. Tests were performed 30 to 90 minutes after sampling. Previous studies have shown that platelet function screening in whole blood within 3 hours of blood collection gives satisfactory results [16].

Whole blood aggregometry was performed using a Chronolog (Havertown, PA) dual-purpose aggregometer by measuring the increase in impedance (in ohms) between two electrodes as aggregation occurred. Aggregation was allowed to proceed for 8 to 12 minutes, and maximum impedance measured after 8 minutes was defined as the maximal aggregation response. The following aggregating agents were used (final concentrations): adenosine diphosphate (20 µmol/L), -thrombin (16 µg/mL), TRAP-6 (10 and 12 µmol/L), and collagen (3 µg/mL). These concentrations were high to ensure that any change in response seen after CPB was not due to a shift in the dose-response curve.

Flow Cytometry
A subset of the study patients (10 to 20 patients) had measurements of platelet activation and adhesion performed using flow cytometry. Whole blood was diluted 1:10 in a modified Tyrode’s buffer (137 mmol/L NaCl, 2.8 mmol/L KCl, 1 mM MgCl₂, 12 mmol/L NaHCO₃, 0.4 mmol/L Na₂HPO₄, 10 mmol/L N-(2-hydroxyethyl)piperazine-N'-2-ethanesulphonic acid, 0.35% bovine serum albumin, and 5.5 mmol/L glucose; pH 7.4) within 10 minutes of the blood drawing, and 45-µL aliquots were incubated for 10 minutes at room temperature with either 5 µL of buffer or agonist. The agonists used were -thrombin (2 U/mL) or TRAP-6 (12 µmol/L). The peptide GPRP was added to all -thrombin samples to prevent fibrin formation. The samples then were incubated for 20 minutes at room temperature with a saturating concentration of the appropriate antibodies.

For dual-color platelet activation analysis in whole blood, the monoclonal antibody CD-42b-PE, specific for platelets (glycoprotein Ib/IX), was added to all samples. In addition, one of the following was used: PAC-1-FITC, specific for activated platelets (glycoprotein IIb/IIIa activation complex); S-12-FITC, specific for platelet P-selectin; IgG-FITC, used as a control for S-12; or IgM-FITC, used as a control for PAC-1.

Samples for tricolor platelet–white blood cell (WBC) interaction analyses were prepared by diluting citrated whole blood 3:7 with the modified Tyrode’s buffer. Aliquots (100 µL) were incubated for 10 minutes at room temperature with either an agonist or buffer. The following monoclonal antibodies were used: CD-45-PerCP, specific for all WBCs; CD-14-FITC, specific for all monocytes; and either CD-42b-PE, specific for all platelets, or IgG-PE, used as a control for CD-42b-PE. All samples were fixed with 1% paraformaldehyde for 60 minutes at room temperature, stored at 4°C, and processed within 24 hours. All antibodies that required conjugation with a fluorescent marker (PAC-1 or S-12) were titrated before use.

Samples were analyzed by a FACScan Flow Cytometer (Becton-Dickinson, Mountain View, CA) using Becton-Dickinson’s Cell Quest software for analysis.

Platelets in dual-color samples were identified by the characteristic forward and side scatter pattern of PE-labeled events. Activation of the platelets was measured by the percentage of platelets that expressed the green fluorescence of the PAC-1 or S-12 antibody above that of their respective controls and the mean fluorescence intensity (MFI; in arbitrary fluorescence units) of the total platelet population. Each sample analyzed 10,000 platelet events.

White blood cells with adherent platelets in whole blood were analyzed by a tricolor method that first identified the three WBC populations (monocytes, lymphocytes, and polymorphonuclear leukocytes) and then measured the PE-positive events (specific for platelets) within these populations (Fig 1). The identification of these three populations was accomplished by collecting all events that were positive for CD-45-PerCP (all WBCs) and placing electronic bit maps (or gates) around populations that were (1) positive for CD-14-FITC (monocytes), (2) partly positive for CD-14-FITC (polymorphonuclear leukocytes), and (3) negative for CD-14-FITC (lymphocytes). Fig 1A shows a representative dot-plot obtained using tricolor flow cytometry. The percentage of WBCs with adherent platelets was determined by measuring the percentage of events positive for CD-42b-PE (platelets) within each WBC population above that of the IgG-PE control. Fig 1B shows representative histograms of platelet events in the polymorphonuclear leukocyte population for 1 patient, using TRAP as the agonist.

View larger version (29K): [in this window] [in a new window]   Tricolor flow cytometry results for thrombin receptor agonist peptide (TRAP)–induced platelet adhesion to polymorphonuclear leukocytes (PMNs). (A) The three leukocyte populations. (B) TRAP-stimulated platelet populations adherent to PMNs before, immediately after, or 24 hours after cardiopulmonary bypass. (FITC = fluorescein isothiocyanate.)

Statistics
Whole blood aggregation response to the various aggregating agents before, 30 minutes after, and 24 hours after CPB were compared using repeated-measures analysis of variance with corrected t-tests for intergroup comparisons. Platelet aggregation responses in the high- and low-transfusion groups were compared using the Mann-Whitney rank-sum test. Flow cytometry parameters in the three treatment groups (before, 30 minutes after, and 24 hours after CPB) were compared by repeated-measures analysis of variance with adjusted t tests for intergroup comparisons. Probability values of 0.05 or less were considered statistically significant.

	Results

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Whole Blood Aggregation
The whole blood aggregation response in 79 patients during CPB is shown in Fig 2. The solid areas of the bars represent the 95th percentile above or below the mean, the error bars represent 2 standard deviations above or below the mean, and the waist in the solid area represents the mean value for each aggregating agent at each time interval. The mean TRAP-induced whole blood aggregation response decreased significantly both immediately after (6.5 ± 1.6 ) and 24 hours after (5.6 ± 1.4 ) CPB compared with preoperative values (12.8 ± 1.5 ; p < 0.01). These were the only statistically significant differences for any of the aggregating agents at any of the time points.

View larger version (39K): [in this window] [in a new window]   Whole blood aggregation responses to various aggregating agents before (pre-op), immediately after (immed p-op), and 24 hours after (24 h p-op) cardiopulmonary bypass. (ADP = adenosine diphosphate; TRAP = thrombin receptor agonist peptide.)

View larger version (31K): [in this window] [in a new window]   Thrombin receptor agonist peptide (TRAP)–induced whole blood (WB) aggregation response 24 hours after cardiopulmonary bypass (24th post-op) in patients who required excessive blood transfusion (white bars) compared with those who did not require excessive blood transfusion (dark bars).

View larger version (24K): [in this window] [in a new window]   Thrombin receptor agonist peptide (TRAP)–induced expression of activated fibrinogen receptor on platelets during cardiopulmonary bypass. (Immed post-op = immediately after operation; pre-op = before operation; 24th post-op = 24 hours after operation.)

Effect of Cardiopulmonary Bypass on Platelet–White Blood Cell Adhesion (Tricolor Flow Cytometry)
Leukocytes are thought to have a modulating effect on platelet function, and platelets express surface molecules that facilitate leukocyte–platelet adhesion. Tricolor flow cytometry was used to measure agonist-induced platelet–leukocyte adhesion in response to TRAP and a nonspecific leukocyte stimulant, formyl-methionyleucyl-phenylalanine (used as a control). Fig 5 summarizes the results of these experiments. The percentage of polymorphonuclear leukocytes that contained adherent TRAP-stimulated platelets was decreased significantly immediately after CPB (37% ± 12%; p < 0.001) compared with preoperative values (76% ± 22%) and values obtained 24 hours after CPB (64% ± 18%).

View larger version (34K): [in this window] [in a new window]   Alterations in thrombin receptor agonist peptide (TRAP)–induced platelet–leukocyte adhesion during cardiopulmonary bypass (CPB). (FMLP = formyl-methionyleucyl-phenylalanine; PMNs = polymorphonuclear leukocytes; post-CPB = after cardiopulmonary bypass; pre-op = before operation; 24th p-op = 24 hours after operation.)

	Comment

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

The platelet whole blood aggregation response to TRAP, but not to thrombin, is decreased after CPB (see Fig 2). This divergence of results between two agonists that presumably stimulate the same receptor deserves further discussion. Recently, a putative second thrombin receptor has been identified using "knockout" mice that are deficient in the gene for the conventional thrombin receptor originally cloned in Coughlin’s laboratory [4] [25] [26]. The second thrombin receptor identified in knockout mice is inferred from studies using TRAPs. The platelets in knockout mice are normally responsive to thrombin, but their fibroblasts do not respond either to TRAPs or to thrombin [25]. This is strong inferential evidence that a second thrombin receptor exists on mouse platelets. Our results, shown in Fig 2, support the concept of a second thrombin receptor, distinct from the STTR, on human platelets. The thrombin responsiveness of the putative second thrombin receptor seems to be relatively protected from the damaging effects of CPB. Further studies are required to elaborate on this preliminary finding.

Increased blood use is associated with CPB-induced STTR desensitization. This suggests that the generation of thrombin and perhaps other proteolytic enzymes during CPB may be responsible for desensitizing the STTR and compromising hemostasis after operation. This thrombin-related hemostatic defect involves at least two steps: (1) generation of thrombin (eg, from extrinsic pathway activation through tissue factor and factor VII [1]), and (2) desensitization or incomplete activation of the STTR on target cells such as platelets and endothelial cells. It would follow that strategies aimed at limiting the harmful effects of thrombin during CPB would result in improved hemostasis.

Drugs or antibodies that are STTR antagonists may have clinical benefit in limiting postoperative blood loss. It is reasonable to suggest that trials aimed at protecting the STTR during CPB, perhaps using hirudin or other STTR antagonists in conjunction with heparin, are indicated to limit blood transfusion in high-risk patients [27] [28].

Leukocytes are thought to modulate platelet function, and various soluble mediators play a role in this regulation [29]. For example, thrombin-induced platelet activation is downregulated by neutrophils in vitro [29]. Activated platelets translocate adhesion molecules to their surface that facilitate the binding of WBCs. One of these adhesion molecules is P-selectin. Blockade of P-selectin–mediated binding of platelets to WBCs results in downregulation of thrombin-induced platelet activation [29]. We observed a significant decrease in the expression of P-selectin on TRAP-activated platelets after CPB. Simultaneously, we found a very significant decrease in platelet–polymorphonuclear leukocyte binding in response to TRAP after CPB (Fig 5). Likewise, Rinder and co-workers [30] [31] found that CPB induces the loss of platelet adhesion receptors, the formation of platelet–leukocyte conjugates, and the activation of leukocytes. Our studies suggest that abnormalities in TRAP-induced platelet functions, including platelet–leukocyte adhesion, are correlated with excessive postoperative blood transfusion. Taken together, these results suggest that thrombin-induced platelet leukocyte adhesion contributes to hemostasis, especially after CPB.

The adhesion molecule or molecules responsible for this decreased agonist-induced platelet–leukocyte binding is uncertain. However, because platelet P-selectin is known to induce platelet–leukocyte adhesion, and in view of our results using a specific monoclonal antibody directed at platelet P-selectin, it seems likely that P-selectin–mediated platelet–leukocyte adhesion is at least partly responsible for the observed effect. Other adhesion molecules also may play a part in this effect. Furthermore, our results and those of others [28] suggest a procoagulant role for thrombin-induced P-selectin–mediated binding of platelets to leukocytes (and perhaps endothelial cells). Further work needs to be done to characterize the significance of multicellular (especially platelet–leukocyte) modulation of molecular processes during CPB and their effect on postoperative hemostasis.

In summary, platelet responses to TRAP are decreased after CPB, and this defect is associated with increased postoperative blood transfusion. This suggests that the generation of thrombin and resultant activation of the thrombin receptor are important causes of postoperative bleeding. Attempts to control blood loss during CPB, especially in high-risk patients, should include the protection of the thrombin receptor and limitation of thrombin generation during operation.

	Acknowledgments

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
We thank Mrs Ruth Myer for her expert help in the preparation of the manuscript.

	References

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 

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