D-Amino Acids in Peptides from Animals, Including Human: Occurrence, Structure, Bioactivity and Pharmacology.

All life forms typically possess homochirality, with rare exceptions. In the case of peptides and proteins, only L-amino acids are known to be encoded by genes. Nevertheless, D-amino acids have been identified in a variety of peptides, synthesized by animal cells. They include neuroexcitatory and neuroprotective peptides, cardioexcitatory peptides, hyperglycemic hormones, opioid peptides, antimicrobial peptides, natriuretic and defensin-like peptides, and fibrinopeptides. This article is a review of their occurrence, structure and bioactivity. It further explores the pharmacology and potential medical applications of some of the peptides.

Delayed thrombin generation is not associated with fibrinopeptide formation during prolonged cardiopulmonary bypass with hirudin anticoagulation.

Patients with heparin-induced thrombocytopenia urgently requiring surgery with cardiopulmonary bypass (CPB) present a unique management challenge that must be addressed by the use of alternative anticoagulants. Although clinical success with the direct thrombin inhibitor hirudin has been reported, there is sparse information in the literature supporting the efficacy of this drug as an anti-thrombotic to prevent fibrin formation during CPB. In this report, we describe the efficacy of this drug to prevent thrombin-mediated fibrin formation during CPB.

Increased incidence of neoplasia of the digestive tract in men with persistent activation of the coagulant pathway.

BACKGROUND: Thrombin promotes angiogenesis and cell proliferation in cancer. Whether thrombin turnover influences cancer incidence is unknown. OBJECTIVES: To explore the relation between the status of the coagulant pathway and cancer incidence by population survey. METHODS: Of 4,009 middle-aged men clinically free of malignancy, 3052 (76.1%) were recruited. Measurements of hemostatic status were made annually for 4 years, and follow-up for morbidity and mortality was maintained thereafter. Persistent activation of the coagulant pathway was diagnosed when prothrombin fragment 1+2 and fibrinopeptide A concentrations exceeded the upper quartiles of the population distribution in two consecutive annual examinations. Cancer incidence rates in men developing persistent activation (taking the time of onset of activation as baseline) were compared with those in men remaining free of this condition. RESULTS: Persistent activation of the hemostatic pathway was a distinct entity found in 111 men [43 expected by chance alone (P <0.001)], and associated with activation throughout the coagulation pathway. Total mortality (/1000 person-years) was higher in those with persistent activation than in others (17.1 and 9.7, respectively, P=0.015), owing to a higher mortality from all cancers (11.3 and 5.1, respectively, P=0.01), due in turn largely to a higher mortality from cancers of the digestive tract (6.3 and 1.9, respectively, P=0.004). Trends were similar for non-fatal cancers. CONCLUSIONS: Persistent activation of the coagulant pathway plays a role in the preclinical phase of cancer and is associated with an increased incidence of clinical malignancy, especially of the digestive tract.

Influence of different anticoagulant agents on fibrinopeptide a generation.

The purpose of this study was to determine the in vitro effects of different anticoagulant drugs on fibrinopeptide A (FPA) generation inhibition and to identify whether there is any correlation between FPA generation, Hemochron ACT, global clotting assays, and chromogenic assays. Unfractionated heparin is a conventionally used anticoagulant. New anticoagulant drugs such as low molecular weight heparins (LMWHs), pentasaccharide, and antithrombin drugs are now approved for various indications. Anti-Xa drugs are in various phases of clinical development. The influence of different anticoagulant agents has been studied on fibrinopeptide A generation, Hemochron celite ACT, global clotting assays, and chromogenic anti-Xa and anti-IIa assays. Different LMWHs (Clivarin, Dalteparin, Enoxaparin, and Tinzaparin), anti-Xa agents (Pentasaccharide, DX-9065a and unfractionated heparin), and anti-IIa agents (PEG-Hirudin, Hirudin, Efegatran and Argatroban) were studied. The blood from healthy volunteers (n=4) was drawn for each drug. Imuclone FPA enzyme-linked immunosorbent kit assay, Hemochron celite ACT assay, global clotting assays (PT, APTT, Heptest-HI, thrombin time), and Loyola chromogenic anti-Xa and anti-IIIa assays were studied. Pentasaccharide demonstrated minimal effects on the whole blood clotting time such as ACT and on inhibition of FPA generation (IC50 > 25 microg/mL). DX-9065a exhibited a significant prolongation of ACT and marked inhibition of FPA generation (IC50 = 4.12 microg/mL). Unfractionated heparin showed a marked inhibition of FPA generation (IC50 = 5.16 microg/mL). Pentasaccharide, DX-9065a and UFH showed a marked correlation between ACT and inhibition of FPA generation. LMWHs demonstrated concentration-dependent inhibition of FPA generation. LMWHs studied showed good correlation between FPA generation inhibition and ACT test. Similar correlation was seen between FPA generation inhibition and the APTT, anti Xa (heptest-HI assay) and anti-IIa activity. Anti-IIa drugs demonstrated concentration-dependent inhibition of FPA generation. Their FPA generation inhibition potency is correlated with the ACT assay. A strong correlation between Hemochron ACT and FPA generation inhibition was observed. Based on this significant correlation, the FPA generation inhibition can be predicted by point-of-care ACT assay.

Effects of a synthetic factor Xa inhibitor (JTV-803) on various laboratory tests.

Synthetic direct inhibitors of factor Xa are capable of prolonging the global anticoagulant assay times in a concentration-dependent fashion. The relative degree of thrombin generation inhibition at an equivalent prolongation is not similar to the results observed with heparins and oral anticoagulant drugs. In addition, the direct factor Xa inhibitors prolong the Russell's viper venom test (RWT) and Heptest clotting times. Ecarin clotting time (ECT) and thrombin time (TT) remain unaffected. The kinetics of factor Xa inhibition are markedly different than those observed with pentasaccharide and heparins. Therefore, the methods developed for heparins and pentasaccharides may not be applicable for the monitoring of factor Xa inhibitors. To test the feasibility of using the prothrombin time (PT), International Normalized Ratio (INR), activated partial thromboplastin time (aPTT), Heptest, thrombin time, RVVT, ECT, and a modified anti-Xa amidolytic assay, to monitor a synthetic factor Xa inhibitor, normal human pool plasma samples were spiked with a synthetic factor Xa inhibitor in the concentration range of 0 to 1 microg/mL and 0 to 25 microg/mL. Different laboratory tests were performed and INR and other ratios were calculated. The anticoagulant effects on whole blood were measured using the activated clotting time (ACT). Further studies on the effect of factor Xa inhibitor on platelet aggregation; factor II, VII, and X functional levels; and fibrinopeptide A (FPA) generation were carried out at equivalent INR levels in comparison to oral anticoagulant and antithrombin agents. FPA generation at equivalent anticoagulant level in comparison to heparin (twice the baseline) was also carried out. Factor Xa inhibitor produced a concentration-dependent prolongation of the ACT. ACT was doubled at a concentration of 4 to 5 microg/mL. There was a marked difference in the prolongation of the PT by a synthetic factor Xa inhibitor dependent on the ISI of the PT reagent used. When the results were calculated to determine INR, marked variations were noted between the recombinant thromboplastin and rabbit brain thromboplastin. The rabbit brain thromboplastin reagent gave markedly high INR values. Similar results were observed when different aPTT reagents were studied. In the anti-Xa assay, modification of the incubation time was employed to extend the proper sensitivity range. These studies warrant further investigation to understand the mechanism of action of factor Xa inhibitors.

Hemostasis and fibrinolysis in patients with intermittent claudication: effects of prostaglandin E1.

There is evidence that the coagulation system is activated in patients with peripheral arterial occlusive disease (PAOD). The beneficial effects of the vasoactive drug prostaglandin E1 (PGE1) may rely in part on the modulation of the coagulation system. The study was designed to evaluate the effects of PGE1 on hemostatic and fibrinolytic variables in patients with intermittent claudication. Therefore molecular markers of thrombin (prothrombin fragment 1+2, PTF 1+2; thrombin-antithrombin III complexes, TAT) and fibrin formation (fibrinopeptide A, FPA) and markers of the fibrinolytic activity (fibrin degradation products, D-dimers) were determined before and immediately after the first PGE1 dose (60 microg in 100 ml NaCl over 2 h i.v.) as well as after 4 weeks of daily infusion therapy in 12 PAOD patients and in eight control patients before and after a single placebo infusion. Plasma levels of PTF1+2, TAT, FPA and D-dimers tended to decrease after the initial dose of PGE1. Infusion therapy with PGE1 for 4 weeks led to a decrease of all hemostatic and fibrinolytic parameters with most pronounced changes for PFT1+2, D-dimers and plasminogen activator inhibitor-1 decreasing by 11% (P<0.05), 20% (P<0.05), and 7% (P<0.05), respectively. These variables remained unchanged in controls with placebo infusion. In summary, infusion therapy with PGE1 in patients with PAOD reduces thrombin formation and results in a decrease of fibrin degradation. PGE1 may thus reduce fibrin deposition involved in the pathogenesis of atherosclerosis.

Aspirin potentiates LPS-induced fibrin formation (FPA) and TNF-alpha-synthesis in whole blood.

The effect of aspirin on LPS-incubation of whole blood was investigated. Aspirin induced a concentration dependent increase (2.5-5-fold at 5 mM aspirin) in LPS-induced appearance of TNF-alpha and fibrinopeptide A (FPA) in plasma, despite the concomitant increase in the inhibitory cytokine IL-100. Aspirin substantially raised the levels of LPS-induced TF-mRNA and TNFalpha-mRNA in monocytes isolated from whole blood. The median ratio for TF-/beta-actin mRNA increased from 1.5 +/- 0.44 in the presence of LPS-alone, to 2.5 +/- 0.51 when 5 mM aspirin was added. The TNFalpha/beta-actin mRNA ratios were 1.8 +/- 0.4 and 5.5 +/- 2.7 respectively. Addition of exogenous PGE2 before incubation nearly abrogated the effect of aspirin on TNF-alpha, substantiating the role of PGE2 as a regulator of TNF-alpha synthesis, whereas the effect on FPA was small. Thus, in the presence of LPS in this whole blood model, aspirin apparently had a pro-inflammatory rather than an anti-inflammatory effect.

Effects of recombinant human soluble thrombomodulin (rhs-TM) on clot-induced coagulation in human plasma.

Recent studies have suggested that clot-bound thrombin plays an important role in thrombus growth. In this study, we examined the effects of recombinant human soluble thrombomodulin (rhsTM) on clot-induced coagulation. rhsTM enhanced the activation of protein C by clots, and attenuated clot-induced thrombin generation and fibrinopeptide A (FPA) production in a dose-dependent manner. The inhibitory effect of rhsTM was abolished by anti-protein C antibody. The inhibitory effect of rhsTM on clot-induced thrombin generation continued for over 60 min after the addition of the clot, while an active site-directed thrombin inhibitor, argatroban, produced a more transient inhibition. rhsTM also inhibited the regrowth of the clot in (125)I-fibrinogen-supplemented plasma. We also examined the effect of rhsTM by thromboelastography, rhsTM reduced the growth of the clot but had little effect on the time to begin clotting, while heparin and Fragmin (low molecular weight heparin) had effects opposite to those of rhsTM. These findings suggest that rhs-TM attenuates the growth of the clot by activating protein C and inhibiting further thrombin generation in the clot.

A novel nucleotide-based thrombin inhibitor inhibits clot-bound thrombin and reduces arterial platelet thrombus formation.

A novel thrombin inhibitor based on single-stranded (ss) deoxynucleotides with the sequence GGTTGGTGTGGTTGG (thrombin aptamer) has been recently discovered. In this study, we tested its efficacy in inhibiting clot-bound thrombin activity and platelet thrombus formation in an ex vivo whole artery angioplasty model. The thrombin aptamer showed a specific dose-dependent inhibition of thrombin-induced platelet aggregation (0.5 U/mL) in human platelet-rich plasma, with an IC50 of approximately 70 to 80 nmol/L. In an in vitro clot-bound thrombin assay system, heparin, used at clinically relevant concentrations of 0.2 U/mL and 0.4 U/mL, was ineffective in inhibiting clot-bound thrombin (6.5% and 34.9% inhibition at 0.2 U/mL and 0.4 U/mL, respectively). In contrast, the thrombin aptamer at an equivalent anticoagulant concentration inhibited clot-bound thrombin (79.7% inhibition). In an ex vivo whole artery angioplasty model, the thrombin aptamer markedly suppressed the generation of fibrinopeptide A (FPA), whereas heparin at 2 U/mL was ineffective. Compared with a scrambled ssDNA control, the thrombin aptamer reduced platelet deposition by 34.5% +/- 5% (mean +/- SEM, n = 4, P = .09) at low shear rates (approximately 200 s-1) and 61.3% +/- 11% (mean +/- SEM, n = 4, P = .05) at high shear rates (approximately 850 s-1). Thrombin aptamers based on ssDNA molecules represent a new class of thrombin inhibitors with potent anticoagulant and antithrombotic properties.

Treatment of uremic anemia with recombinant erythropoietin also reduces the defects in platelet adhesion and aggregation caused by uremic plasma.

In the present study, uremic patients on chronic maintenance hemodialysis were treated with recombinant erythropoietin. Before and after 20 weeks of treatment, platelet adhesion and aggregation were studied with perfusions over a sprayed collagen surface and over matrix of cultured endothelial cells with high tissue factor activity. The influence of the erythropoietin induced raise in hematocrit on platelet transport and adhesion was excluded by performing the perfusions at a standard red blood cell concentration. The present study clearly demonstrates that erythropoietin treatment improves platelet adhesion and aggregation in addition to and independent of its effect on the hematocrit. Studies with control platelets resuspended in plasma of untreated patients showed that a uremic plasma factor reduced adhesion and thrombin- and collagen-dependent aggregation. Patient platelets resuspended in control plasma showed no defects. After erythropoietin treatment, the plasma-induced inhibition of adhesion and aggregation had almost completely disappeared from patient plasma. The beneficial effect of the erythropoietin treatment on uremic hemostasis is therefore twofold. The increase of the red blood cell mass improves transport of platelets, and thus adhesion to the vessel wall. The intrinsic defect due to the presence of an inhibitory toxin in uremic plasma is, in large part, corrected. Improved neutralization of uremic toxins by red blood cells or less production of toxins by better oxygenated tissue might play a role in the observed phenomena.

The anticoagulant effect in heparinized blood and plasma resulting from interactions with extrinsic pathway inhibitor.

The influence of Extrinsic pathway inhibitor (EPI) on global clotting times of plasma was studied using activity-blocking IgG antibodies. Dilute tissue thromboplastin (TP) clotting times in plasma collected after intravenous injection of heparin were dramatically shortened by the addition of anti-EPI IgG. Anti-EPI IgG shortened the TP times to a lesser degree in plasma heparinized in vitro. Compared to plasma heparinized in vitro, the TP clotting times were markedly prolonged in post-heparin plasma of equal heparin concentration. Addition of anti-antithrombin IgG reduced the clotting times somewhat more than did anti-EPI IgG, particularly in normal plasma. In plasma from patients with cancer, about equal effect was obtained by blocking either EPI or antithrombin. These clotting time studies suggested that much of the anticoagulant effect caused by injection of heparin depended on EPI. This was confirmed by recording the release of fibrinopeptide A (FPA), as marker of thrombin generation, following addition of TP and CaCl2 to citrated blood. Thrombin generation was delayed and markedly reduced in post-heparin blood compared to that in normal blood. After incubating post-heparin citrated blood with anti-EPI IgG, the generation of FPA was more rapid; the amounts released 30 seconds after addition of TP were 6 times greater (36 vs 6 ng/ml) than in post-heparin blood without anti-EPI IgG. The subsequent FPA values were midway between pre-injection and post-heparin values. In conclusion, between one third and one half of the inhibition of TP-initiated coagulation in post-heparin plasma depends on EPI. This inhibition is mainly due to inactivation of the factor VIIa-TP complex. A small, but distinct contributing effect observed in the APTT assay (and hence no TP) indicates that even increased inactivation of activated factor X contributes. In cancer patients, these EPI-heparin interactions contribute even more to the anticoagulant effects of heparin.

Coordinate regulation of fibrinogen subunit messenger RNA levels by glucocorticoids in primary cultures of Xenopus liver parenchymal cells.

Fibrinogen synthesis is specifically induced by a synthetic glucocorticoid, dexamethasone, in primary liver parenchymal cell cultures of the frog Xenopus laevis. Here we demonstrate that this increase in the level of fibrinogen protein production is accompanied by an induction in the three mRNAs coding for the fibrinogen subunits, designated A alpha, B beta, and gamma. The stimulation of fibrinogen mRNA levels appears to be mediated by the glucocorticoid receptor, because 1) the dose-response relationship parallels the reported affinity of dexamethasone for the Xenopus glucocorticoid receptor; and 2) the induction is blocked by RU 486, a potent antiglucocorticoid. All three subunit mRNA levels are induced coordinately by the hormone. The response is characterized by a detectable increase as early as 2-4 h after dexamethasone addition, continuing to a final 10- to 30-fold increase over basal levels by 60 h. The induction is specific for the fibrinogen mRNAs; total cellular RNA content and the levels of other mRNAs are unaffected by the hormone. Dexamethasone-mediated stimulation of A alpha and B beta mRNA production occurs in the absence of protein synthesis, whereas increased production of gamma mRNA is completely blocked under the same conditions. Thus, the A alpha and B beta genes are probably regulated at least in part by direct transcriptional activation by glucocorticoid-receptor complexes. Induction of the gamma gene is dependent on newly synthesized or labile proteins, which could be required for either transcription or posttranscriptional processes. These data suggest that different proteins are involved in regulation of the three fibrinogen genes.

Heparin requires both antithrombin and extrinsic pathway inhibitor for its anticoagulant effect in human blood.

Heparinization of blood inhibited the generation of fibrinopeptide A (FPA) after addition of thromboplastin (TP). Heparinization was more effective when performed in vivo than in vitro; the amounts of FPA at 60 s incubation were 8% and 32%, respectively, of control values in nonheparinized blood. When monospecific, neutralizing IgG against extrinsic pathway inhibitor (anti-EPI) were added to heparinized blood prior to TP, the amount of FPA increased to 65%. When monospecific IgG blocking antithrombin (anti-AT) was used, the amount of FPA increased to values similar to those in nonheparinized blood. When anti-AT and anti-EPI were both added to heparinized blood, FPA was generated about 25% faster than in normal blood. These results show that EPI contributes significantly to the anticoagulant effect of heparin in human blood.

Effect of ritanserin, a 5-hydroxytryptamine2-receptor antagonist, on platelet function and thrombin generation at the site of plug formation in vivo.

To investigate the role of serotonin in platelet plug formation we studied, in eight healthy volunteers, the effect of ritanserin (a 5-hydroxytryptamine2-receptor antagonist) on the platelet release reaction (represented by beta-thromboglobulin release) platelet prostaglandin metabolism (represented by thromboxane B2 formation), and thrombin generation (represented by fibrinopeptide A formation) in the microvasculature. After administration of ritanserin lower amounts of thromboxane B1 were generated in the initial stages of plug formation, suggesting an inhibitory effect on the platelet prostaglandin metabolism. Similar amounts of beta-thromboglobulin were released after the administration of ritanserin compared with placebo, indicating a minor effect of ritanserin on the release reaction. Reduction of thrombin formation by ritanserin in the later stages of hemostasis suggested an inhibitory effect of this substance on the procoagulatory activity of platelets or endothelial cells. This could be attributable to interference with the formation or function of coagulation factor complexes on cell surfaces, or it could be the consequence of a reduction of the platelet activity.

In vitro evaluation of heparin fractions: old vs. new methods.

Conventionally, heparin has been evaluated for its anticoagulant effect by various nonspecific, poorly standardized coagulant methods such as the U.S. Pharmacopeial and other global (APTT) tests. However, these assays are relatively insensitive to heparin fractions and fragments, and give varying with the newer modes of heparin administration. It has become necessary, therefore, to develop other methods able to detect a proper therapeutic/prophylactic level and to evaluate a standard potency for these agents. Newer amidolytic assays utilizing synthetic substrates provide for specific anti-factor Xa or anti-factor IIa evaluation. Although these factors are believed to reflect the antithrombotic effect of heparin and its fractions/fragments, conclusive clinical studies are not available at this time. Interactions with non-AT-III-mediated pathways, the contact, kallikrein-kinin, and fibrinolytic system, other coagulant factors, endothelium, and eicosanoid system all can contribute to the overall antithrombotic response of heparin. Some of these actions are not measurable by current in vitro test methods. Amidolytic, immunochemical, and physical assays using various activators to generate endogenous factor Xa, as well as assays for the detection of metabolic or release products of each of these systems, may be more relevant to in vivo conditions. Specific low molecular weight markers such as fibrinopeptide A, which provide the earliest detection of hemostatic activation, can be measured directly, or an in vitro system can be developed to quantitate the relative generation of these markers. These methods can then be modified to assay the in vitro potency of heparin preparations. With proper consideration of reagent specificity and concentration, molecular interactions, reaction time and temperature, ionic type and concentration, reliable accurate methods for a true in vitro representation of in vivo events may be developed. Clinical assays must measure an endogenous response to therapy and not merely an absolute circulating level of drug. However, a standardized pure enzyme system may be ideal to evaluate the potency of these agents. The development of heparin derivatives necessitates a reassessment of currently practiced laboratory technology. Furthermore, well-defined biochemical methods are required for a standardized evaluation of potency and clinical monitoring of these agents.

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)

Blood coagulation and acute iron toxicity. Reversible iron-induced inactivation of serine proteases in vitro.

Coagulopathy is a hallmark of severe ferrous sulfate poisoning in humans and laboratory animals. Although nontransferrin-bound Fe3+ is thought to initiate the disorder, little is known about how it interferes with blood coagulation. At iron concentrations comparable to those of previous animal investigations, we reproduced the coagulopathy, in other words, the dose-related prolongation of the prothrombin, thrombin, and partial thromboplastin time, in human plasma in vitro. Studies of the mechanism by which iron prevents a normal plasma coagulation revealed that the proenzymes of the coagulation cascade and fibrinogen were not damaged by iron. Fibrinogen coagulability and fibrin monomer aggregation were unaffected by very high iron concentrations. Instead, thrombin was markedly inhibited by iron in its clotting effect on fibrinogen and, specifically, in its fibrinopeptide A-generating capacity, the inhibitory effect being reversible upon iron removal by EDTA chelation and gel filtration. Thrombin generation in the presence of iron was reduced as well, indicating an inhibition of one or several other enzymes of the intrinsic coagulation cascade. Because the amidolytic activity of human thrombin as well as factor Xa, kallikrein, and bovine trypsin was also reversibly suppressed by ferrous sulfate as well as ferric citrate, we consider it likely that the coagulopathy occurring in iron poisoning is the consequence of a general, physiologically important phenomenon: the susceptibility of serine proteases to nontransferrin-bound Fe3+.