Thrombosis is local coagulation or clotting of the blood in a part of the circulatory system. A more balanced clot is called a thrombus, must develop a seal which is long lasting on large damaged blood vessels. The clotting procedure is self-restricting and anti-thrombin prevent the clot from continuous enlargement, otherwise the developed clot block the blood flow in the vessel. Thrombosis (excessive blood clot formation) within healthy blood vessels can be harmful if not treated within time. Cardiovascular diseases like heart attack, stroke and pulmonary embolism and several others have been related to inappropriate blood clot formation Furie and Furie, 2008. Synthetic drugs show many side effects and require closed monitoring e.g., tranexamic acid (anti-fibrinolytic drugs) and vitamin K has been used in the treatment of bleeding disorder Sànchez-Lamar et al., 1999.

Platelets have important role in blood haemostasis as well as in pathogenesis of cardiovascular diseases Campbell et al., 2008Elliott et al., 1997. Platelet aggregation blood test (PABT) checks how well platelets, clump collectively and cause blood to become a clot Elliott et al., 1997. Blood clot is the aggregation of platelets, red blood cells and fibrin formation through a specific pathway that helps maintain hemostasis. When injury of blood vessel wall occurs, clot formation is completed within 5 minutes in a healthy person Sànchez-Lamar et al., 1999.

Chilgoza pine nuts (Pinus gerardiana) are the edible seed of the Chilgoza pine plant which belong to pinaceae family commonly found in Pakistan, India, China and Afghanistan Destaillats et al., 2010. P. gerardiana nuts contain un-saturated fatty acids which are very beneficial for lowering high cholesterol level. P. gerardiana nuts are good source of energy (628 kcal) and nutrition consisting of protein (11.6g/100g), carbohydrates (19.3g/100g) and fatty acids (61g/100g) Brufau et al., 2006. Regarding nutrition, P. gerardiana nuts contain vitamins, beta-carotene, thiamin (B1), riboflavon (B2), niacin (B3), pantothenic acid (B5), vitamin B6, folate (B9), vitamin C, vitamin E, vitamin k and minerals including calcium, iron, magnesium, manganese, phosphorus, zinc, potassium and phosphorus other constituents including water, so it can be used for both curative & nutritional purposes Sagrero-Nieves, 1992Savage, 2001.

P. gerardiana nut oil has anti-bacterial, anti-fungal, anti-viral, anti-septic, anti-neuralgic, choleretic, diuretic, expectorant, hypertensive properties Amr and Abeer, 2011Lawlees, 1992. Simillarly, hydro-alcoholic extract showed anti-inflammatory anti-bacterial and antioxidant properties Sharma et al., 2016.

Since oxidative stress contributes significantly in platelet activation Bartimoccia et al., 2015 and also synthetic anti-inflammatory drugs like aspirin has antiplatelet activity Roth and Majerus, 1975, the objectives of this study was to investigate the fibrinolytic activity and effect on platelet aggregation.

Platelets are blood cells that contribute in primary hemostasis. Primary hemostatic plug is formed by platelet-platelet interaction. Many studies have been carried out to develop anti-thrombotic agents with improved efficacy for preventing or treating arterial and venous thrombosis. The fibrinolytic enzyme prevents formation of fibrin clots in circulatory system. Some medicines like urokinase and streptokinase are widely used to inhibit homeostatic disorders, particularly thrombo emboli. The primary function of fibrinolytic activity is to disperse fibrin clot in a circulation. Alpha-2-anti-plasmin, plasminogen and tissue plasminogen activators all of which also play a vital role in clot lysis Biggs, 1972Li et al., 2010.

Current study describes the effect of pinus gerardiana nut concentrate (PGCN) on fibrinolytic activity and platelet aggregation in-vitro and in-vivo. P.gerardiana nut oil has increased the lysis of clot in comparison to control. Blood cells from fibrinolytic activity was observed by using a spectrophotometer ( Figure 1A ) The heme released from haemoglobin of red blood cells when 0.1N hydrochloric acid (HCl) was added to the solution of the red blood cells and then acid haematin was formed ( Figure 1B ). The release of RBCs and acid haematin were observed by spectrophotometry. In one of the study aqueous Rue extract was found effective as fibrinolytic effect by 12.53±5.67%. In our study, the fibrinolytic effect of P. gerardiana was found to be increased by 47% and 72%. A number of plant extracts and their products having fibrinolytic activity are recognized included Lumbricus rubellus, Pleurotus ostreatus, Spirodela polyrhiza, Flammulina velutipes, and Ganoderma lucidum, Ginger (Zingiber officinale), Garlic (Allium sativum) as well as from Bacillus sp. in Korean and Japanese fermented foods, chung kookjang, respectively Michelson et al., 2006.

The interactions between platelets and a variety of adhesive proteins, such as collagen, and soluble agonists, such as ADP supply potential targets for developing anti-platelet agents. Different therapies are available to prevent irregular activation and aggregation of platelets. Epinephrine is an adrenergic agonist, bind with alpha 2-adrenergic receptors on platelets and also increases the effect of aggregation caused by other platelet agonists Porth, 2005Strukova, 2001. The capability of epinephrine induces the effect of aggregating agents on accumulation like fibrinogen binding, intracellular Ca²⁺ recruitment, granular relief and protein phosphorylation, and there are some factors correlate with platelet hyper-activity and resulting untimely coagulation of blood Choi, 2002Lanza et al., 1988. Similarly, collagen is the most thrombogenic component of the sub-endothelium Baumgartner and Haudenschild, 1972.

Following vascular damage, collagen is exposed to circulating platelets and both acts as a substrate for the adhesion of platelets Cowan et al., 1981Poole and Watson, 1995 and induces platelet activation Poole and Watson, 1995.

The main evidence suggests that two receptors are involved in the platelet response to collagen, integrin α2β1 acts to adhere platelets to collagen, allowing platelets to interact with the lower affinity receptor glycoprotein VI, which is mainly responsible for platelet activation Morton et al., 1989Santoro et al., 1991.

At lower concentrations, many of the effects of collagen are enhanced by its production of thromboxane A2 (TXA) Nakano et al., 1986Pollock et al., 1986Rittenhouse and Allen, 1982. The collagen-induced increase in [Ca²⁺] can be decreased by inhibiting the production of TXA via the pretreatment of platelets with cyclooxygenase inhibitors such as aspirin Nakano et al., 1986.

P. gerardiana nuts oil concentrate by using its different concentrations was tested for platelet aggregation in the presence of both epinephrine and collagen showed opposite effect on platelet aggregation time ( Figure 2A,B ). Previously unrefined methanolic extract of Nepeta juncea were examined for action against human platelet and have shown significant inhibitory effects on platelet aggregation Hussain et al., 2009. Also it is investigated that extracts of garlic, onions, nettle and alfalfa are proved to be the most potent inhibitors of platelet aggregation in vitro Pierre et al., 2005. Camomile, bramble are similarly powerful at repressing platelet accumulation in-vitro Pierre et al., 2005. Toona microcarpa Harms leaf (TMHE) extract in-vitro significantly inhibited platelet aggregation induced by thrombin, but not by ADP or collagen Choi, 2002. Parsley extract inhibits in vitro and ex vivo platelet aggregation and prolongs bleeding time in rats Mekhfi et al., 2006. In this study, opposite effects of PGNC could be due to specific interference of phytochemicals with collagen or epinephrine dependent cell signaling in platelets.

Hemostasis is a process that is divided into two stages: platelet aggregation and coagulation. P. gerardiana nuts oil increased clotting time in comparison to control which was induced by calcium chloride ( Figure 3 ). This suggests that PGNC can be used cardiovascular diseases for anticoagulation. In-vivo effects of PGNC on blood indices were analysised. The result showed that P. gerardiana had no effect on CBC parameters. Result interpreted that there was no significant effect on RBCS, WBC, HGB, HCT and other parameter. P. gerardiana nuts oil has slightly increased the effect of MCHC but other parameters did not show any significant effect.

In summary, our data strongly suggest that P. gerardiana nut oil can be used in the treatment of thromboembolic disorders and this protective mechanism is associated with increased fibrinolysis and inhibition of collagen-stimulated platelet aggregation.

CBC: complete blood count; Coll: collagen; EDTA: ethylene-diamine-tetra-acetic acid; EPN: epinephrine; HCT: hematocrit; HGB: hemoglobin; MCH: mean corpuscular hemoglobin; MCV: mean corpuscular volume; PGNC: Pinus gerardiana nuts oil concentrates; PLT: Platelets; PRP: platelet rich plasma; RBC: red blood cells; WBC: white blood cells.

AUR give the concept, designed experiment, analyzed data and gave final approval of the manuscript and was the supervisor of the project. SN performed experiment, analyzed data and wrote draft of manuscript. MZ helped in data acquisition and experiment conditions optimization SSH and JA helped in editing and literature review for the paper AAZ did data acquisition, organization and data analysis and helped in write up of final draft.