Pentadecapeptide BPC 157 Reduces Bleeding and Thrombocytopenia after Amputation in Rats Treated with Heparin, Warfarin, L-NAME and L-Arginine

Background BPC 157 is a stable gastric pentadecapeptide recently implicated with a role in hemostasis. While NO is largely implicated in hemostatic mechanisms, in tail-amputation-models under heparin- and warfarin-administration, both the NO-synthase (NOS)-blocker, L-NAME (prothrombotic) and the NOS-substrate L-arginine (antithrombotic), were little investigated. Objective. To investigate the effect of L-NAME and L-arginine on hemostatic parameters, and to reveal the effects of BPC 157 on the L-NAME- and L-arginine-induced hemostatic actions under different pathological condition: tail amputation without or with anticoagulants, heparin or warfarin. Methods Tail amputation, and/or i.v.-heparin (10 mg/kg), i.g.-warfarin (1.5 mg/kg/day for 3 days) were used in rats. Treatment includes BPC 157, L-NAME, L-arginine, per se and their combination. Results After (tail) amputation, with or without i.v.-heparin or i.g.-warfarin, BPC 157 (10 μg/kg, 10 ng/kg, i.p., i.v. (heparin), 10 μg/kg i.g. (warfarin)) always reduced bleeding time and/or haemorrhage and counteracted thrombocytopenia. As for L-NAME and/or L-arginine, we noted: L-arginine (100 mg/kg i.p.)–rats: more bleeding, less/no thrombocytopenia; L-NAME (5 mg/kg i.p.)-rats: less bleeding (amputation only), but present thrombocytopenia; L-NAME+L-arginine-rats also exhibited thrombocytopenia: L-NAME counteracted L-arginine-increased bleeding, L-arginine did not counteract L-NAME-thrombocytopenia. All animals receiving BPC 157 in addition (BPC 157μg+L-NAME; BPC 157μg+L-arginine, BPC 157μg+L-NAME+L-arginine), exhibited decreased haemorrhage and markedly counteracted thrombocytopenia. Conclusions L-NAME (thrombocytopenia), L-arginine (increased haemorrhage) counteraction and BPC 157 (decreased haemorrhage, counteracted thrombocytopenia) with rescue against two different anticoagulants, implicate a BPC 157 modulatory and balancing role with rescued NO-hemostatic mechanisms.


Introduction
This study further extends and clarifies the effect of the stable gastric pentadecapeptide BPC 157 on hemostasis [1,2] with addition of N(G)-nitro-L-arginine methylester (L-NAME) and Larginine and heparin and warfarin in rats.
Thus, it was likely that after tail amputation in rats, with/out heparin and warfarin administration, BPC 157 would counteract the consequences of administration of the NOS-blocker, L-NAME (i.e., prothrombotic effect and thereby thrombocytopenia) and/or NOS-substrate, Larginine (i.e., antithrombotic effect and thereby more bleeding) [29][30][31].

Materials and Methods Animals
Male Albino Wistar rats were used in all of the experiments (10 rats per experimental group and interval). The study was approved by the Local Ethics Committee at School of Medicine (University of Zagreb, Zagreb, Croatia) and experiments were assessed by observers unaware of the given treatment.

Bleeding procedures, heparin, warfarin application and medication
Bleeding was monitored as described before [1], until blood flow stopped for a complete 30-s interval or till the end of 60 min period. In deeply anaesthetised rats (placed in ventral position) the tail was transected with a surgical scalpel 3 cm from the tip (and submerged into a tube with 15 ml of saline at room temperature in vertical position) and the duration and amount of bleeding were measured to evaluate the hemostatic effect of the agents or saline administration. The blood samples assessment was carried precisely as described before [1] (Table 1). In addition, platelet counts were corrected with hematocrit to avoid errors that may be caused by animals bleeding, and were expressed as a percentage of baseline for each time point of the tested groups. To obtain the hematocrit-corrected platelet count (X) we used the following formula: X = (h1xp1 / HxP) x 100, wherein each symbol denotes the following: h1: hematocrit of the sample, p1: platelet count of the sample, H: mean hematocrit value of the normal range in healthy animals (= 0.435), P: mean platelet count value of the normal range in healthy animals (= 875). HxP result was considered as a baseline of 100%.

Amputation associated with warfarin administration
Warfarin was given intragastrically (1.5 mg/kg) once daily for 3 consecutive days, with the last challenge at 3 hours before the bleeding procedure. BPC 157 (10 μg/kg, 10 ng/kg) was given immediately after any warfarin challenge, intragastrically while controls received simultaneously an equivolume of saline (5.0 ml/kg intragastrically). We applied L-NAME (5 mg/kg), L-arginine (100 mg/kg) given per se or combined with each other, intraperitoneally, at 30 minutes before amputation.

Statistical analyses
Statistical analyses of the quantified data were performed by analysis of variance (ANOVA). Post-hoc comparisons were appraised using the conservative Bonferroni/Dunn test. Data are presented as the mean ± standard deviation (SD). Values of P<0.05 were considered statistically significant.

Results
Here, in normal rats, after tail amputation (spontaneous bleeding for 20 minutes, fall in platelet count without any failure of coagulation parameters) the previous effects of BPC 157 (reduced bleeding, no thrombocytopenia) [15] were confronted with that of L-arginine (prolonged bleeding without thrombocytopenia) and L-NAME (reduced bleeding, thrombocytopenia present), given alone and/or combined. Then, these effects were confronted with administration of anticoagulants without or with amputation; heparin (extensive bleeding and blood loss, prominent fall in platelet count, drastic prolongation of PT-, APTT-, TT-values) or warfarin (extensive bleeding and blood loss, prominent fall in platelet count, drastic prolongation of PT-, APTT-values) ( Table 2, Table 3 and Table 4). These effects were specifically assessed as follows (in addition, platelet counts were additionally corrected with hematocrit to avoid errors that may be caused by animals bleeding (Fig 1)).
L-arginine-rats, L-NAME-rats, L-NAME+L-arginine-rats Alone, L-arginine prolonged bleeding time (rats with amputation only) and increased the amount of bleeding (heparin-rats, warfarin-rats) while L-NAME reduced bleeding time (rats with amputation only). L-NAME+L-arginine (combined) exhibited a decrease in bleeding amount and laboratory values in comparison to controls (amputation only) or control-heparin and control-warfarin groups. Commonly, controls (amputation only, heparin-rats and warfarin-rats) exhibited thrombocytopenia. L-arginine counteracted thrombocytopenia (L-argininerats with amputation only and L-arginine-heparin-rats are without thrombocytopenia, L-arginine-warfarin-rats are with thrombocytopenia). Contrary to this, L-NAME-rats or L-NAME +L-arginine-rats consistently exhibited thrombocytopenia.
BPC 157+L-arginine, BPC 157+L-NAME, BPC 157+L-arginine+L-NAME BPC 157+L-arginine-rats BPC 157+L-arginine-rats exhibited shorter bleeding times than corresponding controls after amputation only; amount of bleeding being even less than that of control-heparin-rats or control-warfarin-rats, in addition to attenuated thrombocytopenia after warfarin administration and counteracted thrombocytopenia after amputation only, or amputation and heparin-or warfarin-administration.
BPC 157+L-NAME rats BPC 157+L-NAME rats exhibited shorter bleeding times with lesser bleeding amounts than corresponding controls after amputation only; consistently no thrombocytopenia (amputation only) or attenuated thrombocytopenia in heparin-and warfarin-rats, along with lesser bleeding amounts than corresponding control-heparin-and control-warfarin-rats.
Also, for the consistently reduced amount of bleeding in BPC 157-rats [1,2], the illustrative insight might be the reduced bleeding time evidence, affected by platelet function. Thus, resolving a hemostatic defect, the hallmark of which is a markedly prolonged bleeding time, BPC 157 reduced the bleeding times in the tail amputation model, heparin and partly warfarin rats, but unlike in normal rats, did not reduce bleeding time in heparin and warfarin rats when previously L-arginine or L-NAME was given. Therefore, with respect to BPC 157, L-arginine and L-NAME, heparin and warfarin, we demonstrated a complex and particular interaction.
In the normal rats, L-NAME reduced bleeding time (and induced thrombocytopenia and thereby, the NOS-blocker-prothrombotic effect), L-arginine prolonged bleeding time (thereby showing the NOS-substrate-antithrombotic effect) [29][30][31]. Consequently, changing these effects, we likely revealed, a particular action of L-arginine and a particular action of L-NAME on heparin and warfarin, thus far not demonstrated in amputation rat models. With respect to BPC 157, they might both augment heparin and warfarin effects and thereby BPC 157 would Tail amputation, bleeding time, amount of bleeding, PLT, HCT, PT, APTT, TT, FIB values in warfarin rats. Warfarin was given intragastrically (1.5 mg/kg) once daily for 3 consecutive days, with the last challenge was at 3 hours before the bleeding procedure. BPC 157 (10 μg/kg, 10 ng/kg) was given immediately after any warfarin challenge, intragastrically while controls received simultaneously an equivolume of saline (5.0 ml/kg intragastrically). We applied L-NAME (5 mg/kg), L-arginine (100 mg/kg) alone or combined, intraperitoneally, at 30 minutes before amputation. Mean ± SD, *P<0.05 at least vs. control. have a changed effect (however, as emphasized, BPC 157 always reduced the amount of bleeding). Or, more likely, it could be that their effects are both interfering with that of BPC 157. Likely, this might also be the supportive part to the general interaction. Namely, very similar interactions of BPC 157 and the NO system, with both L-NAME and L-arginine, have been demonstrated by a large number of other previous studies [14,15,[18][19][20][21][22][23][24][25][26]. For instance, counteraction of both the L-arginine-induced disturbance and L-NAME-induced disturbance was seen, while maintaining blood pressure against both L-arginine (hypotension) and L-NAME (hypertension) (note, BPC 157 by itself does not affect basal blood pressure values [25] and likewise, BPC 157 by itself does not affect basal coagulation/platelets values [1]). Thus, this analogy might be the principal advantage for this and previous studies [14,15,[18][19][20][21][22][23][24][25][26], with a challenge in methodology and in general, since in other studies (not related to this model) L-NAME-thrombocytopenia antagonization was not attempted [3][4][5][6][7][8][9]32], nor were L-arginine and L-NAME simultaneously investigated. Furthermore, we could argue that with the prolonged bleeding time, specifically disturbed coagulation factors, with heparin (less) and warfarin (more), would affect BPC 157, L-arginine and L-NAME. Or, more likely, their effects are in general more resistant to that of heparin and less to that of warfarin, regardless of the more haemorrhage in heparin rats.
Illustratively, heparin still induced the full effectiveness of BPC 157 in both doses of application (μg, ng); thrombocytopenia was present only with L-NAME. L-arginine rats did not exhibit thrombocytopenia. Hematocrit-corrected platelet counts (X) before, and after bleeding period. Hematocrit-corrected platelet counts (X) before, and after bleeding period: (a) in normal rats, BPC 157 (10 μg/kg, 10 ng/kg), L-NAME (5 mg/kg), L-arginine (100 mg/kg) given alone and/or together,applied intraperitoneally, at 30 minutes before amputation while controls received simultaneously an equivolume of saline (5ml/kg intraperitoneally); (b) in heparin (10 mg/kg intravenously) challenged rats when treated with BPC 157 (10 μg/ kg, 10 ng/kg), L-NAME (5 mg/kg), L-arginine (100 mg/kg) alone or combined, intravenously, immediately after intravenous heparin while controls received simultaneously an equivolume of saline (0.5 ml/kg intravenously); (c) in warfarin rats (warfarin given intragastrically (1.5 mg/kg) once daily for 3 consecutive days, with the last challenge was at 3 hours before the bleeding procedure). BPC 157 (10 μg/kg, 10 ng/kg) was given immediately after any warfarin challenge, intragastrically while controls received simultaneously an equivolume of saline (5.0 ml/kg intragastrically). We applied L-NAME (5 mg/kg), L-arginine (100 mg/kg) alone or combined, intraperitoneally, at 30 minutes before amputation. Mean ± SD, *P<0.05 at least vs. control, 10 rats at least per group. Contrary to this, particular with warfarin are: a lesser effectiveness of BPC 157 (smaller dose not effective on bleeding time, but still affects amount of bleeding and thrombocytopenia); not only with L-NAME-rats, but also those treated with L-arginine there was an initial marginal thrombocytopenia up to a very prominent thrombocytopenia after amputation, and both exhibited unaffected warfarin-bleeding amounts. Thus, with warfarin, thrombocytopenia is the common end result of the L-NAME and/or L-arginine administration. In consequence, with the thrombocytopenia and haemorrhage, the expected lack of NO-suppression of platelet aggregation [33], the acceleration and amplification of the induced pattern of platelet activation resulting in platelet exhaustion [9] (L-NAME), grossly correspond to the suggested inhibition of platelet aggregation, by direct action on an intraplatelet constitutive calcium-dependent NOS [33][34][35] (L-arginine). In either case, particularly considering the sustained warfarin bleeding, even when L-arginine actually attenuated thrombocytopenia, the disturbed NO-hemostatic system [27,30,31] is the entity which has to be further rescued. Thereby, it is important that thrombocytopenia appeared also in warfarin L-NAME+L-arginine-rats and that it is attenuated by the addition of BPC 157 (L-NAME+L-arginine+BPC 157-rats) and reduced the amount of bleeding. Likewise, the addition of BPC 157 to the L-NAME (L-NAME+BPC 157-rats) or to the L-arginine (L-arginine+BPC 157-rats), equally reduced the amount of bleeding. Thereby, we argue that the better hemostasis was achieved with BPC 157 administration, the balanced role of the L-arginine and of the L-NAME and the more rescued NO-hemostatic mechanisms.
The supportive argument may be that BPC 157 by itself induces NO-release (demonstrated from gastric mucosa supernatant), like L-arginine, but also in conditions where L-arginine is not working [14].
Consequently, whatever the fundamental mechanism of BPC 157 beneficial effects, there was a BPC 157-induced counteraction of the two different anticoagulants and of the L-NAME and the L-arginine. Together, these counteractions could obviously suggest an alternative common pathway.