Laurence A. Sherman

Paradoxic elevation of fibrinopeptide A after streptokinase: Evidence for continued thrombosis despite intense fibrinolysis

Elevated levels of fibrinopeptide A, a marker of thrombin activity associated with acute myocardial infarction, have been found to decrease after administration of streptokinase when reperfusion occurs. In contrast, in patients without reperfusion and those with reocclusion after streptokinase therapy, fibrinopeptide A remains elevated. In the present study early serial measurements of fibrinopeptide A were used to further characterize this paradoxic increase in thrombin activity after streptokinase and to characterize its response to heparin. In 19 patients with acute myocardial infarction fibrinopeptide A was elevated to 82.3 +/- 43.5 ng/ml (mean +/- SE) before therapy. Thirty minutes after the initiation of streptokinase, fibrinopeptide A increased to 300.1 +/- 117.4 ng/ml (p less than 0.01), consistent with extensive thrombin activity. Fibrinopeptide A remained elevated until 15 minutes after a heparin bolus injection when levels decreased to 15% of the poststreptokinase value (49.2 +/- 13.3 ng/ml) (p less than 0.001). These data document a prompt paradoxic increase in thrombin activity after administration of streptokinase that may be responsible for failure of therapy in some patients.

Fibrinopeptide A: a marker of acute coronary thrombosis.

To determine whether coronary thrombosis in vivo is reflected by elevations in levels of fibrinopeptide A (FPA) in plasma, we sequentially characterized plasma FPA levels associated with evolving infarction in patients admitted to the cardiac care unit early after the onset of symptoms, in patients with transmural infarction admitted later, and in patients with nontransmural infarction. Studies were also performed in patients in whom the diagnosis of infarction was suspected but subsequently excluded. FPA values were significantly higher in patients with transmural infarction (42.3 +/- 11.2 ng/ml [mean +/- SEM], n = 53) compared with those in patients with nontransmural infarction (4.8 +/- 1.6 ng/ml, n = 17) or with those in patients in whom infarction was subsequently excluded as a diagnosis (3.5 +/- 0.6 ng/ml, n = 17, p less than .01 for both). Elevations in FPA level were greatest in patients with transmural infarction from whom samples were obtained soon after the onset of symptoms. Thus, in 39 patients from samples were obtained within 10 hr after the onset of symptoms, FPA levels were significantly higher than in 14 patients from whom samples were obtained initially more than 10 hr after the onset of symptoms (55.5 +/- 14.7 vs 4.9 +/- 1.4 ng/ml, p less than .01). In 30 of the 39 patients with evolving transmural infarction from whom samples were obtained within the first 10 hr after the onset of symptoms, the level of FPA was greater than 8 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Importance of continued activation of thrombin reflected by fibrinopeptide A to the efficacy of thrombolysis

Factors responsible for initial success or failure of coronary thrombolysis and persistent recanalization or early reocclusion have not been thoroughly elucidated. Both adequate initial clot lysis and preclusion of rethrombosis are required. Failure may reflect clot lysis followed immediately or somewhat later by rethrombosis. To determine whether differences in the intensity and persistence of the activation of thrombin are determinants of success or failure of recanalization, plasma fibrinopeptide A, a fibrinogen product liberated by thrombin, was serially assayed in 19 patients treated with intravenous streptokinase. In patients exhibiting recanalization (n = 9), plasma fibrinopeptide A decreased after administration of streptokinase but before administration of heparin. In patients without initially apparent recanalization, fibrinopeptide A increased, suggesting ongoing thrombosis, and subsequently decreased promptly after heparin. In patients with initial recanalization followed by overt reocclusion the pattern was different. Despite recanalization, fibrinopeptide A continued to rise markedly. Elevations persisted despite administration of heparin. Thus, inhibition of activation of thrombin is associated with successful recanalization. Conversely, persistent activation of thrombin may be a predisposing factor to both apparent initial failure of recanalization and overt early reocclusion.

Indium‐III: a New Radionuclide Label for Studying Human Platelet Kinetics

Summary. Indium‐III, when complexed with 8‐hydroxyquinoline (oxine), has been employed as a radioactive platelet label for thrombus imaging in animals and man. The short half‐life (2.8 d) and high yield of gamma photons of 111In make it ideal for in vitro counting and external imaging. To evaluate its suitability for studies of platelet turnover in man, platelet kinetic studies were carried out on 10 healthy volunteers using 111In‐and 51Cr‐platelets concurrently. For 111In labelling, platelets were harvested by differential centrifugation from 43 ml of whole blood drawn into acid‐citrate dextrose (ACD) solution. The platelets were washed and suspended in a mixture of ACD and isotonic saline and then incubated with 111In‐oxine, rewashed, and suspended in plasma for reinfusion. 51Cr labelling was performed using standard methods. Mean labelling efficiency was 73% with 111In and 6.5% with 51Cr. In vitro studies demonstrated minimal release, elution, and reutilization of the 111In label. There was no significant difference in the aggregation response of 111In‐ and 51Cr‐platelets to ADP and collagen. The in vivo recovery of 111In‐platelets was approximately 50% greater than that of 51Cr‐platelets whereas the platelet life spans were similar. These results indicate that 111In labelled platelets may be useful for thrombokinetic studies in man. The new method offers the advantages of reduced blood requirements, higher labelling efficiency, and the ability to perform external imaging of platelet distribution in vivo.

Studies of radioiodinated fibrinogen I. Physicochemical properties of the ICl, chloramine-T, and electrolytic reaction products

Abstract The rate of catabolism of fibrinogen is known to be increased by extensive iodination. (MacFarlane, A.S., (1963) J. Clin. Invest. 42, 346–361) Clottability measurements are, however, insensitive to this biological damage. (Regoeczi, E., (1971) J. Nucl. Biol. Med. 15, 37–41). We have used gel permeation chromatography in an attempt to understand the chemical modifications of the protein that underlie this alteration in biological activity. In a comparative study of 125I-labelled fibrinogen labelled by the ICl, chloramine-T, and electrolytic methods, we have found that product 125I-labelled fibrinogen is formed with a molecular weight significantly greater than that of normal fibrinogen. The chloramine-T method gives the highest degree of aggregation and the ICl method gives a product whose molecular weight profile most clearly resembles that of authentic fibrinogen. All of the products are hydrolyzed at similar rates in saline (30 ± 3% in the first 24 h) and in plasma or albumin (2.0 ± 0.5% per day). Some chloramine-T preparations have been noted to be more rapidly hydrolyzed in plasma following a two-day induction period. The rate of breakdown is slower in polyethylene containers than in glass. Our results suggest that ICl preparations of 125I-labelled fibrinogen show the least alteration in chemical properties and may therefore be the best for use in in vivo tracer studies involving radioiodinated fibrinogen.

Relationship between elevated plasma levels of crosslinked fibrin degradation products (XL-FDP) and the clinical presentation of patients with myocardial infarction

To assess whether the intense thrombotic state known to occur early after the onset of acute myocardial infarction is further exacerbated by impaired intrinsic fibrinolysis, we compared the intensity of fibrinolysis as measured by the level of crosslinked fibrin degradation products (XL-FDP) in plasma with the intensity of thrombosis as assessed by fibrinopeptide A (FPA) in 98 patients with transmural and 14 patients with non-Q wave infarction. Patients without complications of infarction such as shock, mural thrombi, or malignant arrhythmias requiring countershock generally had normal plasma levels of XL-FDP, less than or equal to 300 ng/ml (81% of those presenting less than 8 hours after onset and 66% of those presenting greater than 8 hours after onset) on admission despite elevated FPA indicative of ongoing thrombosis. In contrast, patients with complications generally had elevated levels of XL-FDP greater than 300 ng/ml (80% of those presenting early and 62.5% of those presenting late) and 50% of these patients had marked elevations to greater than 1000 ng/ml. FPA was markedly elevated in patients with complications whether they presented early or late after onset of infarction. Our direct measurements at the time of infarction support previous data indicating that intrinsic fibrinolysis is impaired in patients with acute infarction, despite marked thrombin activity, when complications are not present. However, when complications are present initially, a more exuberant fibrinolytic response is observed perhaps due to thrombosis associated with the complications themselves.

Macromolecular complexes formed as the result of chloramine-T radioiodination of proteins

Fibrlnogen has recentiy been shown to form macromolecular aggregates during radioiodination with chloramine-T. Decreased half-lives of chloramine-T radioiodinated fibrinogen have also been noted. The effects of chloramine-T on fibrinogen fragment D, fibrinopeptide A, albumin and gamma G globulin were studied to determine if aggregation is unique to fibrinogen. With fragment D and albumin, ICl-labelled protein had identical column behavior to the unlabelled protelns. Chloramine-T labelled protein showed protein products of higher molecular weight than native Fg-D. Albumin was reacted with varylng amounts of chloramine-T in the absence of iodide. In high C-T concentrations, there was significant production of higher molecular weight species. These results suggest that use of standard chloramine- T radioiodination may result in macromolecular species which would be unsuitable for in vlvo purposes. It would seem desirable that any protein labelled with chloramine-T for in vivo studies should be examined for the presence of this type of subtle denaturation prior to use. (auth)

Investigation of an inflammatory humoral factor as a stimulator of fibrinogen synthesis

A purported stimulatory effect on fibrinogen synthesis of pyrogenic crude leukocyte extract (CLE) is controversial. To clarify this issue, a study was made of possible inflammatory cellular or humoral factors as stimulators of fibrinogen synthesis. Relative fibrinogen synthesis ratios were calculated by comparing the rate of incorporation of radiolabeled lysine into fibrinogen with that of incorporation into albumin. Significant increases in the ratio over baseline values were observed in rabbits after i. v. administration of CLE derived from 0.99 – 3. 35×109 homologous leukocytes harvested from inflammed rabbit peritoneal cavities (0.146±0.038 control and 0.249±0.119 after CLE, p<0.01). Wash fluid which was used to collect the peritoneal leukocytes was also infused and found to increase ratios above baseline (0.118±0.052 control and 0.226±0.094 after inflammed peritoneal, wash fluid). This difference, however, was only borderline statistically significant (p = 0.05). In contrast, no significant increases above baseline were observed in the ratio following intravenous administration of 1) blood leukocyte derived CLE, 2) intact blood leukocytes, or 3) plasma, all harvested from turpentine or glycogen injected animals. These data suggest that at inflammatory sites, granulocytes produce a mediator(s) which increases fibrinogen synthesis during inflammation. It is well known that during many types of inflammation plasma fibrinogen concentration increases. However, little is known of the mechanisms which mediate increased fibrinogen synthesis during inflammation. Pyrogenic substances released from rabbit granulocytes have been reported to cause increases in serum acute phase proteins in recipient rats (1–3). These factors, variably called leukocyte endogenous mediator (LEM), leukocyte pyrogen (LP), or crude leukocyte extract (CLE), have also been reported by Bocci (4) to result in an increase in plasma fibrinogen concentration over controls when given in “significant” amounts to homologous recipient rabbits. On the other hand, Seligsohn (5), while using a similar homologous rabbit model, failed to demonstrate a significant increase in plasma fibrinogen concentration or synthesis following CLE administration. With this controversy in mind, we investigated the possible existence of a cellular or humoral stimulator of fibrinogen synthesis during inflammatory states in a homologous rabbit system. As potential sources of mediator, we sampled materials from a site of inflammation. These were crude leukocyte extract derived from leukocytes and wash fluid from the peritoneum after sterile peritonitis was induced in rabbits. In addition, we sampled materials distant from the site of inflammation. These were peripheral blood, buffy-coat leukocytes and plasma as well as crude leukocyte extract derived from buffy-coat leukocytes. The effect of the infusion of these homologous experimental materials on fibrinogen synthesis was measured by determining the relative fibrinogen synthesis rate as adapted from that described by Koj (6). With this technique, the amount of incorporation of radiolabeled lysine into fibrinogen is compared to that of its incorporation into albumin during the same measurement period. Albumin is used since it is a non-inflammatory protein (6). The relative synthesis rate of fibrinogen (fg) versus albumin (alb), or fg/alb ratio, approximates the ratio of the absolute synthesis rate of the two proteins. Unlike techniques following 15Se-methionine incorporation into fibrinogen (7), this method allows sequential use of 3H and 14C-lysine permitting both a baseline control and experimental study in the same animal. Increases above control of this ratio indicates an increase in synthesis rate of fibrinogen. This method also has advantages over measuring changes in fibrinogen concentration, because it is faster and more sensitive.

Quantitation of the Reticuloendothelial System Clearance of Soluble Fibrin

Phagocytosis of particulate fibrin has been previously established as a function of the reticuloendothelial system (RES). More recently, reticuloendothelial cells have been shown to bind soluble fibrin/fibrinogen (f/F) complexes in vitro. To quantitate RES clearance us microthrombus formation, varying doses (0.1‐6 mg/kg) of 125I‐soluble f/F was injected into rabbits. One hour later the animals were killed, at which time 48

The 125i lAbeled Fibrinogen Test in the Diagnosis of Postoperative Thrombophlebitis

pM 8% of the 125I f/F had been cleared from the blood. Homogenized organ samples were separated into insoluble pellet, soluble protein bound, and free 125I. Treatment of other samples with plasmin prior to homogenization differentiated the insoluble 125I into RES cleared (intracellular‐plasmin resistant) vs microthrombi (plasmin sensitive pellet 125I). 125I‐f/F was chiefly found in liver and spleen. Injection of low f/F concentrations resulted in no plasmin sensitive pellet 125I. 3 mg/kg f/F caused small, variable amounts of plasmin sensitive pellet 125I, chiefly in the kidney. With 6 mg/kg, 21‐50% of the insoluble 125I in all organs was plasmin sensitive, and occasional 1–2 mm thrombi were found. The data indicate complete and rapid RES clearance of small amounts of soluble f/F from the blood, without microthrombi being formed. The RES was acutely saturated at 1.5–3.0 mg f/F/kg, which is equivalent to immediate conversion to fibrin of 1–2% of the intravascular fibrinogen pool.